Team:Hong Kong-CUHK/Laboratory/log book

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Revision as of 15:20, 5 October 2011

2011 CUHK iGEM LOG BOOK

➨ Select week number from the table above...

Week 0

2 June2011 (Tue)

1. Frankie- transformed the bacteria with RFP and prepared the T7 and HR gene plasmid for PCR.

Week 1 Monday 6 June – Sunday 12 June

8 June2011 (Wed)

1. Test the effect of different concentration of NaCl to the survival of bacterial DH5a& BL21

9 June2011 (Thu)

1. Repeat Wed work

2. IPTG inducer

3. PCR amplification - T7 and HR(rhodopsin)

4. Gel clean to get T7 and HR gene from gel

10 June2011 (Fri)

1. Restriction cut of T7, HR and iGEM vector, ligate them together

2. Received all medium to grow magnetobacteria

Week 2 Monday 13 June – Sunday 19 June

13 June 2011 (Mon)

1. Nanodrop test DNA concentration

2. Transformation to competent cells, spread on plate A & plate K

14 June 2011 (Tue)

1. no colonies were found on spread plate. possible reasons:

Ø antibiotic C resistance spread on plate K

Ø failed restriction cut,

Ø spreading tools too hot which kill the bacteria…

2. Attempt to mix culturing medium for halobacterium salinarum DSM 3754, haloterrigena turkmenica DSM 5511, Natronomonas pharaohs DSM 2160, but couldn't find casamino acids which are essential for all the medium required

3. Repeat work of last week: restriction cut, ligation

15 June 2011 (Wed)

1. transformation of E.coli competent cells.

Week 3 Monday 20 June – Sunday 26 June

20 June (Mon)

1.      Restriction cut

Ø   pSB1A3 & pSB1K3

Ø   HR & T7 promoter

Ø   restriction enzyme: EcoR1 &Pst1

Ø   incubate at 37^oC , 2hours

2.      PCR purification of cut product

Ø   refer to kit protocol

3.      Agarose gel electrophoresis (Run gel) of purified product

Ø   at 120V, 45minutes

Ø   Failed. Possible reasons: insufficient DNA conc. In gel, stock problem

4.      Ligation

Ø   HR+pSB1A3

Ø   T7+ pSB1A3

Ø   HR+ pSB1K3

Ø   T7+ pSB1K3

Ø   at 16^oC, overnight

5.      Inoculation transformation buffer preparation

Ø   Steps of preparing 500ml inoculation transformation buffer

1.      55mM MaCl₂‧4H₂O (5.4425g)

2.      15mM CaCl₂‧2H₂O (1.1025g)

3.      250mM KCl (9.3189g)

4.      10mM PIPES (10ml) (taken from 4^oC fridge)

5.      Stored in 4^oC fridge

Ø   109 plate with antibiotic A and K is poured

6.      Detection of T7 and HR by gel electrophoresis

Ø   no band of sample is shown

21 June (Tue) No record

22 June (Wed)

1.      Pick colonies

Ø   Each plate pick 2 colonies into 2 tubes

2.      Inoculation

Ø   at 37^oC, 250rpm, 7hours

3.      Transfer

Ø   DH5α(B tube) : 1ml & 0.5 ml

Ø   BL 21 (B tube) : 1ml & 0.5 ml

Ø   Rosetta (A tube): 2ml

Ø   Overnight, 200rpm

4.      Prepare 500ml LB, 500ml LB with 1M NaCl and 500ml LB with 1M KCl solution LB solution

5.      transform pET27bHR with K resistance into DH5α, BL21

23 June (Thu)

1.      Measure OD of transfer

Ø   All tubes’ OD > 0.55

Ø   Due to contamination in the step of picking colonies. We shouldn’t put the pipette tips into the tubes since our medium does not contain antibodies

2.      Repeated the experiment from picking colonies and inoculation

Ø   Only DH5α is used

Ø   7 hours inoculation

Ø   Inoculation failed.

3.      Restarted from culturing E.coli on agar plate

Ø   DH5α & BL21

4.      Restriction cut and Double Digestion

Ø   HR   20ng/ul

Ø   T7   20ng/ul

Ø   Since there are not enough solution(a 50ul system should have at least 200ng of DNA) to make up a 50ul system, we modify the system to 25ul.

Solution (added by order)

Amount(ul)

ddH₂0

14

10X Buffer 3

2.5

10X BSA

2.5

EcoR1

0.5

Pst1

0.5

HR

5

Total

25ul

Solution (added by order)

Amount(ul)

ddH₂0

14

10X Buffer 3

2.5

10X BSA

2.5

EcoR1

0.5

Pst1

0.5

T7

5

Total

25ul

Solution (added by order)

Amount(ul)

ddH₂0

15

10X Buffer 3

2.5

10X BSA

2.5

EcoR1

0.5

Pst1

0.5

pSB1

4

Total

25ul

Ø   The 3tubes are put at 37 ^oC for 2hours

5.      Restriction cut

Ø   T7/HR restriction cut mixture

Ø   pSB1 T3 restriction cut mixture

Ø   The mixture is put into thermomixer at 37oc for 2hs

Ø   Inoculated 5ml start culture of DH5α,, no BL21 colony is observed on the plate

Ø   6 sets of solution is prepared

0 – 1M NaCl with IPTG

0 – 1M NaCl without IPTG

0 – 1M KCl with IPTG

0 – 1M KCl without IPTG

0M salt with IPTG

0M salt without IPTG

(the solution without salt is set as controls)

[Salt]/M

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

LB + salt/ml

0

0.4

0.8

1.2

1.6

2.0

2.4

2.8

3.2

3.6

4

LB/ml

4.0

3.6

3.2

2.8

2.4

2.0

1.6

1.2

0.8

0.4

0

Ø   For each tube, 2ul IPTG is added (for sample with IPTG), 40ul DH5α start culture, 4ml LB+ salt volumes

Ø   5ml start culture of DH5αis inoculated again for the miniprep on next day

Ø   OD of solution is checked on next day

24 June (Fri)

1.      Picking E.coli colonies cultured yesterday

Ø   DH5α & BL21

2.      Inoculation:

Ø   4 DH5α tubes & 4 BL21 tubes

Ø   Each with 5ml LB

Ø   37℃, 250rpm

3.      Transfer inoculation mixture into 125ml LB in conical flask

Ø   1.0ml DH5α-2, 0.5ml DH5α-2

Ø   1.0ml BL21-2, 0.5ml BL21-2

Ø   Shake at 37℃, 250rpm, overnight

4.      Transform “HR+1T3” & “T7+1T3” into DH5α competent cells

Ø   1.0ml HR+1T3, 0.5ml HR+1T3

Ø   1.0ml T7+1T3, 0.5ml T7+1T3

Ø   1 control plate without antibiotics tetracycline

Ø   Incubate at 37℃, overnight

5.      Light absorbance of DH5α KCl IPTG

Ø   DH5α KCl, DH5α NaCl IPTG and DH5α NaCl is measured

Light absorbance of DH5α KCl IPTG

Concentration/M

OD (ABS/cm)

Final OD (ABS/cm) (without dilution)

0.1(5x)

0.532

2.660

0.2(5x)

0.515

2.575

0.3(5x)

0.477

2.385

0.4(5x)

0.390

1.950

0.5(5x)

0.370

1.850

0.6(5x)

0.267

1.335

0.7(5x)

0.92

0.960

0.8(2x)

0.300

0.600

0.9(2x)

0.156

0.312

1.0

0.249

0.249

0

0.791

0.791

Light absorbance of DH5α KCl

Concentration/M

OD (ABS/cm)

Final OD (ABS/cm) (without dilution)

0.1(5x)

0.500

2.500

0.2(5x)

0.447

2.235

0.3(5x)

0.430

2.150

0.4(5x)

0.399

1.995

0.5(5x)

0.305

1.525

0.6(5x)

0.264

1.32

0.7(5x)

0.205

1.025

0.8(5x)

0.117

0.585

0.9(2x)

0.220

0.440

1.0

0.263

0.263

0(5x)

0.519

2.595

Light absorbance of DH5α NaCl IPTG

Concentration/M

OD (ABS/cm)

Final OD (ABS/cm) (without dilution)

0.1(2x)

0.829

1.658

0.2(2x)

0.844

1.688

0.3(5x)

0.898

1.796

0.4(5x)

0.384

1.920

0.5(5x)

0.340

1.700

0.6(5x)

0.208

1.040

0.7(2x)

0.393

0.786

0.8(5x)

0.287

0.574

0.9(2x)

0.119

0.238

1.0

0.113

0.113

Light absorbance of DH5α NaCl

Concentration/M

OD (ABS/cm)

Final OD (ABS/cm) (without dilution)

0.1(5x)

0.499

2.495

0.2(2x)

0.478

2.390

0.3(5x)

0.513

2.565

0.4(5x)

0.390

1.950

0.5(5x)

0.302

1.510

0.6(5x)

0.246

1.230

0.7(5x)

0.194

0.970

0.8(2x)

0.269

0.538

0.9

0.284

0.284

1.0

0.103

0.103

6.      Miniprep

7.      Storage of cells

Ø Transfer 1ml from each cell culture to 1.5ml microcentrifuge tube

Ø Spin for 1min

Ø Remove medium by discarding and pipetting

Ø Store the cells at -80^oc

25 June (Sat)

1.      OD measurement (OD 600nm) of transferred product

BL21

0.5ml

1.891

BL21

1.0ml

1.831

DH5α

0.5ml

0.670

DH5α

1.0ml

1.415

Ø   All tubes’ OD > 0.55

Ø   Might have contamination

Ø   Still used DH5α 0.5ml 0.670 OD600 to make competent cells

Ø   Made 25 tubes of competent cells at -80℃

2.      pick colonies, Inoculation,

Ø   2 from HR+1T3, 2 from T7+1T3 II, 1 from HR+1T3 I into 5ml LB+tetracycline snap-cap tube

3.      Transfer 125ml magnetotactic bacteria medium to a conical flask, waiting for autoclave

Week 4 Monday 27 June – Sunday 3 July

27 June (Mon)

1. Inoculated 5ml start culture of DH5α transformed with HR pET37b using the plate the week before

2. Prepared 6 conical flasks

Ø LB with 50ul IPTG + 100ul antibiotic K

Ø LB without IPTG + 100ul antibiotic K

Ø 0.6M NaCl LB with 50ul IPTG + 100ul antibiotic K

Ø 0.6M NaCl LB without IPTG + 100ul antibiotic K

Ø 0.6M KCl LB with 50ul IPTG + 100ul antibiotic K

Ø 0.6M KCl LB without IPTG + 100ul antibiotic K

Ø For measuring OD absorbance and plot growth curve of DH5α on 28/06/2011 every 30/45min

3. Prepared 4 sets of solution same as 23/06/2011 to verify the optimum concentration of salt for DH5α to grow and absorb salt and added DH5α start culture

4. Inoculate 5ml start culture of DH5α transformed with HR pET27b using the plate of the week before for the 6 conical flasks for 8/06/2011 growth curve (+5ul antibiotic K)

28 June (Tue)

1. Prepare another set of competent cell

Ø Pick single colony from plates prepared on Sat

Ø Transfer the colony into 5ml of LB broth in FALCON

Ø Incubate for 6hrs @37℃, shaking (250-300rpm)

Ø ~6pm, use the incubated starter culture to prepare 2 1L flasks

FLASK1: 0.5ml culture + 125LB

FLASK2: 0.25 ml culture + 125LB

Ø Incubate at 22℃, moderate shaking, overnight

2. Transform competent cells prepared on Sat 25 June

Ø Incubate at 37℃, overnight

3. Plasmid DNA purification

Ø T7, PSB1K3 / HR, PSB1K3

Ø USING Spin MiniPrep Kit and a Microcentrifuge

4. Light absorbance of DH5α KCl IPTG, DH5α KCl, DH5α NaCl IPTG, DH5α NaCl, DH5α 0M LB and DH5α 0M LB IPTG

Light absorbance of DH5α KCl IPTG
Concentration/M	OD (ABS/cm)	Final OD (ABS/cm) (without dilution)
0.1(2x)	0.916	1.832
0.2(2x)	0.756	1.512
0.3(2x)	0.527	1.054
0.4	0.426	0.426
0.5	0.710	0.710
0.6	0.310	0.310
0.7	0.156	0.156
0.8	0.129	0.129
0.9	0.072	0.072
1.0	0.012	0.012

Light absorbance of DH5α KCl
Concentration/M	OD (ABS/cm)	Final OD (ABS/cm) (without dilution)
0.1	0.618	2.472
0.2	0.427	1.708
0.3	0.274	1.096
0.4	0.845	1.690
0.5	0.474	0.948
0.6	0.970	0.970
0.7	0.626	1.252
0.8	0.504	0.504
0.9	0.083	0.083
1.0	-0.011	-0.011

Light absorbance of DH5α NaCl IPTG
Concentration/M	OD (ABS/cm)	Final OD (ABS/cm) (without dilution)
0.1(2x)	0.794	1.588
0.2(2x)	0.716	1.432
0.3(2x)	0.483	0.966
0.4(2x)	0.436	0.872
0.5	0.761	0.761
0.6	0.713	0.713
0.7	0.641	0.641
0.8	0.491	0.491
0.9	0.168	0.168
1.0	0.044	0.044

Light absorbance of DH5α NaCl
Concentration/M	OD (ABS/cm)	Final OD (ABS/cm) (without dilution)
0.1(2x)	0.932	1.864
0.2(2x)	0.719	1.438
0.3(2x)	0.535	1.070
0.4(2x)	0.525	1.050
0.5	0.882	0.882
0.6	0.819	0.819
0.7	0.712	0.712
0.8	0.470	0.470
0.9	0.123	0.123
1.0	0.031	0.031

Light absorbance of DH5α 0M LB
Concentration/M	OD (ABS/cm)	Final OD (ABS/cm) (without dilution)
0.1	0.763	1.526
0.2	0.829	1.658

Ø 0.8ml DH5α is added to start culture solution of the 6 conical flask prepared the day before, the absorbance of solution is measured per 30mins

Time(h)\OD of Solution	LB	LB IPTG	0.6M NaCl LB	0.6M NaCl LB IPTG	0.6M KCl LB	0.6M KCl LB IPTG
0	0.015	0.016	0.028	0.030	0.025	0.047
0.5	0.025	0.024	0.050	0.017	0.041	0.014
1	0.035	0.039	0.077	0.015	0.025	0.029
1.5	0.071	0.080	0.070	0.005	0.036	0.026
2	0.146	0.176	0.111	0.005	0.044	0.044
2.5	0.307	0.338	0.148	0.003	0.063	0.057
3	0.473	0.527	0.262	0.017	0.093	0.093
3.5	0.650	0.669	0.345	0.012	0.134	0.137
4	0.874	0.888	0.473	0.003	0.219	0.210
4.5	10.66	1.128	0.626	0.001	0.323	0.310
5	0.753(2x)	0.800(2x)	0.722	0.001	0.386	0.365

Ø 5ml start culture of DH5α is transformed with HR pET27b (using the plate the week before) is inoculated, 6 conical flasks of solution is prepared same as the 12/06/2011

29 June (Wed)

1. Measure OD of competent cell prepared yesterday

Flask	1	2	3	4
OD 600	1.697	1.844	1.840	1.662

Ø All flasks have OD greater than 0.55, it indicates contamination.

Ø Failed

2. Measure DNA concentration

Ø Use DNA purified yesterday

	260/280	DNA concentration (ng/ul)
T7 pSB1K3 (1)	1.95	32.6
T7 pSB1K3 (2)	1.90	32.2
HR pSB1K3 (1)	2.08	12.2
HR pSB1K3 (2)	2.01	12.3

3. Prepare new competent cells

Ø Transfer 0.1 nd 0.5ml DH5a and BL21 into 125ml LB

4. Double restriction cut of T7 pSB1K3 and HR pSB1K3

Water	11.75ul
Buffer 3	3.5ul
BSA	0.35ul
EcoR1	0.7ul
Pst1	0.7ul
T7 pSB1K3	8ul
Total	25ul

Buffer 3	2.5ul
BSA	0.25ul
EcoR1	0.5ul
Pst1	0.5ul
HR pSB1K3	21.25ul
Total	25ul

Ø Incubate at 37oC for 2hrs

5. Run gel

Ø T7: band at ~150kb

Ø HR: band at ~800kb

6. The absorbance of 6 solutions

Time(h)\OD of Solution	LB	LB IPTG	0.6M NaCl LB	0.6M NaCl LB IPTG	0.6M KCl LB	0.6M KCl LB IPTG
0	0.002	0.023	0.019	0.026	0.001	0.001
0.5	0.006	0.004	0.020	0.032	0.003	0.018
1	0.012	0.022	0.043	0.031	0.011	0.006
1.5	0.023	0.028	0.047	0.021	0.017	0.032
2	0.110	0.151	0.065	0.068	0.025	0.021
2.5	0.279	0.345	0.129	0.075	0.029	0.053
3	0.430	0.455	0.488	0.137	0.068	0.057
3.5	0.591.	0.640	0.230	0.183	0.089	0.112
4	0.748	0.830	0.310	0.264	0.122	0.150
4.5	0.962	1.053	0.445	0.353	0.183	0.265
5	0.747	0.832	0.389	0.310	0.177	0.209
5.5	0.939	10.17	0.448	0.380	0.235	0.261
6	0.764	0.745	0.360	0.316	0.251	0.310

30 June (Thur)

1. Measure OD of 0.1 and 0.05ml of DH5a and BL21 in 125 LB prepared on 29 June

	Volume (ul)	OD 600
DH5a	50	0.352
	100	0.523
BL 21	50	0.349
	100	1.710

Ø DH5a 100ul is taken for making competent cells

Ø Use competent cells made to transform T7 and HR

Ø Streak plates to check if competent cells viable

Ø Total of 8 plates

Ø Incubate at 37^oC overnight

2. Spread plates with transformed competent cells

Ø T7 pSB1K3 in DH5a

Ø HR pSB1K3 in DH5a

Ø Total of 4 plates

3. PCR purification of iGEM plasmid

4. PCR purification of plasmid made

Ø 1K3, 1T3, 1C3

5. Run gel

Ø No result

1 July (Fri)

1. Check plates

Ø All 8 plates shown bacteria growth

Ø All 4 plates shown transformed cells growth

2. Pick colonies

Ø DH5a and competent cells with T71K3 and HR1K3

Ø Total of 12 snap caps

Ø Shake at 250rpm, 37^oC overnight

3. PCR amplification and purification of iGEM psB1K3

4. Run gel

Ø No banding

2 July (Sat)

1. Mini prep

Ø DH5a HR x3

Ø DH5a T7 x3

Ø HR1K3 x3

Ø T71K3 x2

Ø One of the T71K3 didn’t form pallet after centrifugation

2. Digestion

Water	11.2ul
DNA	10ul
Buffer 3	2.5ul
EcoR1	0.5ul
Pst1	0.5ul
BSA	0.25ul
Total	25ul

Ø Incubate at 37^oC, 2hrs

Ø Remaining samples are stored at -20^oC

3. Run gel (with 2 setups)

Well	1	2	3	4	5	6
	ladder	T71K3 (1)	T71K3 (2)	HR(1)	HR(2)	HR(3)

Ø Result: Lane 1 and 3 have bands of ~100-200bp

Land 4 to 6 has bands of 800bp

Well	1	2	3	4	5	6	7
	ladder	T7(1)	T7(2)	T7(3)	HR1K3(1)	HR1K3(2)	HR1K3(3)

Ø Result: Lane 2 to 4 has bands of ~100-200bp

Lane 5 to 7 has bands of ~800bp

3 July (Sun): No record

Week 5 Monday 4 July – Sunday 10 July

4 July (Mon)

1. Pick colonies

Ø BL21, 3 snap caps

Ø Incubate at 37oC, 250rpm

1. Prepare plates with antibiotics

Ø 25 plates with C

Ø 43 plates with K

2. Transfer incubated BL21 to 125ml LB, total of 3 samples, each with 50ul BL21 added

Ø Incubate at37^oC overnight, 150rpm

3. 5ml start culture of DH5α is inoculated and put into the incubator in common room

4. 6 conical flasks is prepared

Ø LB IPTG

Ø 0.6M NaCl LB IPTG

Ø 0.6M KCl LB IPTG

Ø 0.1M – 1M NaCl IPTG

Ø 0.1M – 1M KCl IPTG

Ø LB without IPTG

5 July (Tue)

1. Prepare competent cells

	OD
BL21 (1)	1.958
BL21 (2)	1.918
BL21 (3)	1.907

Ø OD greater than 0.55, repeat the experiment

Ø Using BL21, we incubate cells from picking colony from plates

Ø Culture from tubes are transferred to the flask containing LB at 25^oC overnight, 130 rpm

2. PCR purification of T71K3 plasmid

Ø Using iGEM protocol

Ø Concentration of primers is 100pmol/ul, hence we have to dilute it to 30pmol/ul

3. Run gel using iGEM protocol and product protocol

Ø After PCR amplification

Result: both show bandings beyond 100bp

iGEM protocol shows banding at 2000kb

Ø After PCR purification

Result: iGEM protocol shows banding at 2000kb

4. Inoculation

5. 1ml DH5α start culture solution is added to3 conical flasks prepared on 4/7.

Ø The absorbance and cell density is measured every 30 minutes.

Ø Cell density at 2h and 2.5 h is failed to be measured.

Ø Procedure of measuring cell density

i. 1ml solution is transferred to microcentrifuge tube, centrifuge for 1min and resuspend

ii. 10ul solution is transfer to both side of the hematocytometer and cover with the cover slit

iii. observe the central square with 10x magnification under microscope and count the number of cell

Ø Dilution is required (10fold, 100fold, 1000fold .. ) for higher absorbance

Ø (1OD unit = 109 cell)

Ø Absorbance of 21 tubes is measured.

Light absorbance of DH5α NaCl IPTG( 4times diluted)
Concentration/M	OD (ABS/cm)	Final OD (ABS/cm) (without dilution)
0.1	0.701	2.804
0.2	0.786	3.144
0.3	0.680	2.720
0.4(2x)	0.947	3.788
0.5(2x)	0.603	2.412
0.6(2x)	0.538	2.152
0.7	0.334	1.336
0.8	0.083	0.332
0.9	0.025	0.100
1.0	0.022	0.088

Light absorbance of DH5α KCl IPTG( 4times diluted)
Concentration/M	OD (ABS/cm)	Final OD (ABS/cm) (without dilution)
0.1	0.893	3.572
0.2	0.906	3.624
0.3	0.780	3.120
0.4	0.445	1.780
0.5	0.621	2.484
0.6	0.083	0.332
0.7	0.218	0.872
0.8	0.040	0.160
0.9	0.010	0.040
1.0	0.027	0.108

Ø Light absorbance of 3 solutions

Time(h)\OD of Solution	LB IPTG	0.6M NaCl LB	0.6M KCl LB
0	0.025	0.070	0.054
0.5	0.043	0.065	0.044
1	0.117	0.068	0.067
1.5	0.185	0.041	0.059
2	0.389	0.069	0.064
2.5	0.634	0.085	0.081
3	1.039	0.107	0.111
3.5	1.586	0.188	0.181
4	0.334	0.274	0.268
4.5	0.364	0.350	0.328
5	2.412	0.432	0.450

Ø Cell density (HR, IPTG, LBK, DH5α, pET27b HR)

Time(h)	Averaged cell density
0	1.5 x 10⁴
0.5	2.0 x 10⁴
1	1.3 x 10⁵
1.5	2.0 x 10⁶
2	-
2.5	-
3	8.95 x 10⁶
3.5	8.30 x 10⁹
4	1.145 x 10¹⁰
4.5	6.70 x 10⁹
5	6.10 x 10⁹

6 July (Wed)

1. Prepare BL21 competent cell

BL21	OD
1	1.094
2	1.263
3	1.263
4	1.163

Ø OD>0.55, preparation failed

2. Restriction cut of T71K3 prepared on 5July

Buffer 3	5ul
BSA	0.5ul
Eco R1	1ul
Pst 1	1ul
T71K3	10ul

3. Run gel to check the size of 2 sets of T71K3

Ø Both samples show banding of ~4000kb

4. Streak plate: BL21 X7

5. 5ml start culture of DH5α is inoculated and put into the incubator in common room

6. 3 conical flasks is prepared

Ø LB IPTG

Ø 0.4M NaCl LB IPTG

Ø 0.4M Kcl LB IPTG

7 July (Thu)
1. Pick colony of BL21 X4 and inoculation

Ø 37^oC, 250rpm

2. PCR amplification

Ø “Home-made” HR1K3, “Home-made” T71K3, stock 1K3

Ø Using iGEM protocol

PCR supermix	45ul
Upper primer	1ul
Lower primer	1ul
Water	25ul
DNA	0.5ul
TOTAL	50ul

3. Transfer 50ul BL21 to 125ul LB

Ø Inoculated at 37^oC, 250rpm

4. Run gel

Ø Lane1: ladder

Ø Lane3: “Home-made” HR1K3

Ø Lane4: “Home-made” T71K3

Ø Lane5: stock 1K3

5. OD of BL21 in 125ml LB

	1	2	3	4
16:30	0.006	0.007	0.007	0.020
16:45	0.007	0.013	0.01	0.007
20:45	0.143	0.085	0.085	0.136
22:15	0.367		0.312

6. 5ml DH5α start culture solution is added to3 conical flasks prepared on 5/7

Ø The absorbance and cell density is measured every 30 minutes

7. 3 conical flasks is prepared

Ø 100ml LB + 50ul IPTG + 100ul antibiotic K

Ø 60ml LB + 40ml KCl LB + 50ul IPTG + 100ul antibiotic K

Ø 60ml LB + 40ml NaCl LB + 50ul IPTG + 100ul antibiotic K

Time(h)\OD₆₀₀ of Solution	LB IPTG	0.4M NaCl LB	0.4M KCl LB
0	0.033	0.031	0.025
0.5	0.037	0.032	0.017
1	0.078	0.013	0.033
1.5	0.122	0.056	0.047
2	0.310	0.102	0.096
2.5	0.423	0.157	0.156
3	0.620	0.255	0.256
3.5	0.893	0.398	0.427
4	1.186	0.590	0.621
4.5	1.632	0.859	0.803

Ø Cell density of DH5α, pET27b HR, LB IPTG with K

Time(h)	Averaged cell density
0	3x10⁶
0.5	2.5 x10⁶
1	3.75 x10⁷
1.5	1 x10⁷
2	9.75 x10⁸
2.5	9.15 x10⁸
3	2.43 x10⁹
3.5	2.555 x10⁹
4	2.7875 x10⁹
4.5	2.225 x10⁹

8July (Fri)

1. Streak plate of Bl21 competent cell

Ø incubate at 37^oC

2. transformation of Pwt: BBa_E0044 (Green Fluorescent Protein deviated from jellyfish Aequeora Victoria wild-type GFP with plasmid pSB1A3, in 2011 kit plate 1 well 14G)

Ø 10ul H20 added to well 14G

Ø DNA concentration was measured: 85.1ug/ul

Ø 1ul of DHA was added to DH5a competent cell

Ø 2 tubes of competent cell was used, one being taken from other bb (labeled as DH5a T) and one being prepared by overselves (labeled as DH5a)

Week 6 Monday 11 July – Sunday 17 July

11 July (Mon)
1. iGEM plasmid stock check:

pSB1K3	~ 0ul
pSB1C3	< 1ul
pSB1T3	~4-5ul
pSB1A3	~4-5ul

Ø Insufficient quantity

Ø Bacterial amplification (plasmid+biobrick) of pSB1A3 (taken from iGEM kit plate 1, 1G)

Ø Use competent cell prepared on 30June

Ø Transformation: put pSB1A3 + biobrick into DH5a

Ø Result refered to 13July

12 July(Tue)
1. Nano drop

T7 + 1K3	42.5 ng/ul
HR + 1K3	13.5 ng/ul

Ø pSB1T3 labeled to be 25ng/ul

1. Double digestion

Ø Incubated at 37^oC, 2hours

H2O	19.25ul
Buffer 2	2ul
BSA	2.5ul
Eco R1	0.5ul
Spe 1	0.5ul
T71K3	0.25ul
TOTAL	25ul

H2O	14.25ul
Buffer 2	2.5ul
BSA	0.25ul
Xbal 1	0.5ul
Pst 1	0.5ul
HR1K3	7ul
TOTAL	25ul

H2O	17.25ul
Buffer 2	2.5ul
BSA	0.25ul
Eco R1	0.5ul
Pst 1	0.5ul
pSB1T3	4ul
TOTAL	25ul

Ø Result: yield of product is too low

2. Prepare T plate

3. Bacterial amplification of backbone + biobrick (Continue experiment on 11July)

Ø red colonies observed, planned to do Midi Prep on 13July

4. 1ml DH5α start culture solution is pipetted to each conical flasks

Ø The LB IPTG solution is contaminated, OD and cell count is too high and out of the normal range.

Ø Frankie advised to use 500ml next time

Ø Brian will check if the cell count machine works

Ø Preparation of NaCl, KCl DH5α should include using pipette to draw all medium away to avoid bacteria killed by ice.

Time(h)\OD₆₀₀ of Solution	LB IPTG	0.4M NaCl LB	0.4M KCl LB
0	0.180	0.049	0.025
0.5	0.234	0.021	0.020
1	0.301	0.041	0.024
1.5	0.377	0.047	0.055
2	0.606	0.127	0.114
2.5	0.744	0.209	0.212
3	0.916	0.324	0.331
3.5	1.125	0.472	0.449
4	1.289	0.673	0.725
4.5	1.432	0.881	0.886
5	1.533	10147	1.122
5.5	1.654	1.345	1.364

Ø Cell density of DH5α, pET27b HR, LB IPTG with K

Time(h)	Averaged cell density
0	2.21x10⁷
0.5	8.3 x10⁸
1	7.9 x10⁷
1.5	3.6 x10⁸
2	3.035 x10⁹

13July (Wed)

1. Double digestion (repeat experiment of yesterday)

Ø Modify the volume to minimize the amount of insert(T7, HR)

Buffer 2	2ul
BSA	0.2ul
Eco R1	0.5ul
Spe 1	0.5ul
T71K3	16.8ul
TOTAL	20ul

Buffer 2	2ul
BSA	0.2ul
Xbal 1	0.5ul
Pst 1	0.5ul
HR1K3	16.8ul
TOTAL	20ul

H2O	12.8ul
Buffer 2	2ul
BSA	0.2ul
Eco R1	0.5ul
Pst 1	0.5ul
pSB1T3	4ul
TOTAL	20ul

2. Run gel to check cut products

1	2	3	4	5	6	7	8
blank	HR+1K3	Ladder	T7+1K3	blank	pSB1T3	blank	blank

3. Transformation of HR+1K3, T7+1K3 prepared on 28June

Ø plates spread with antibiotics K, incubate overnight

Ø plates labeled as “HR+1K3 prepared on 28June” and “T71K3 prepared on 28June”

4. Red colonies observed on plates prepared on 11July

Ø Miniprep tomorrow

5. Gel check after double digestion and gel extraction

Ø Lane3: ladder

Ø Lane6: T7 (13.1 ng/ul) ~200kb

Ø Lane9: HR (25.5 ng/ul) ~800kb

Ø Lane12: 1T3 (2.3ng/ul)

Ø Gel result: weak banding

6. Ligation

T4 ligase buffer	2ul
T4 ligase	1ul
T7	8ul
HR	6ul
Vector (pSB1T3)	3ul
TOTAL	20ul

14July (Thu): no record

15July (Fri)

1. Transformation

Ø DH5a competent cell prepared on 30June

Ø 3 way ligation (T7- HR- 1T3)

2. Inoculation

Ø Red colonies on plates prepared on 17July

Ø 2 centrifuge tubes prepared, labeled as red colony1 & 2

Ø Transferred 250ul to 125mL LB (1:500 ratio)

Ø Shake at 37^oC, 250rpm, overnight

Ø Labeled as red col 1&2

16July (Sat)

1. Mini prep

Ø DNA from red colony culture

Ø 4 tubes of DNA obtained

2. Digestion

DNA	10ul
10X Buffer 3	2.5ul
10X BSA	2.5ul
EcoR1	0.6ul
Pst1	0.6ul
H2O	8.8
Total	25ul

Ø Incubate at 37oC for 2hrs

3. Pick colonies of 3-way assemblyDH5a

Ø Incubate at 37^oC, 250rpm

17 July (Sun)

1. Run gel and gel recovery of red colony

2. Mini prep, run gel and gel recovery of 3-way assembly

3. Run gel using digestion product prepared on 16/7 (red colony)

4. Mini prep DNA of bacteria culture (3-way DH5a)

Ø Total of 5 samples of 3-way 1T3 are made, each with 40ul

Ø Stored at -20^oC

Ø 10ul of each is used for digestion and run gel to check size

Water	11.25ul
DNA	10ul
Buffer 3	2.5ul
EcoR1	0.5ul
Pst1	0.5ul
BSA	0.25ul
Total	25ul

Ø Incubate at 37^oC for 2hrs

5. Gel extraction of gel with 1T3 backbone from red conlony

Ø 4 tubes of backbone are made

Ø Store at -20^oC

6. Pick red colony of DH5a

Ø 4tubes of LB snap cap

Ø Incubate at 37^oC, 25rpm

7. Transfer to 125ml LB

Ø Shake at 37^oC overnight, 250rpm

8. Streak plate using red colony

Ø Plates with antibiotics A are used

Ø Store in incubator

Week 7 Monday 18 July – Sunday 24 July

18 July (Mon)

1. Nanodrop

3-way	260/280	ng/ul
1T3 (1)	1.88	56.3
1T3 (2)	1.91	71.5
1T3 (3)	1.91	33.5
1T3 (4)	1.96	20.8
1T3 (5)	1.87	30.1

1T3 backbone (1)	3.78	3.9
1T3 backbone (2)	1.91	5.8
1T3 backbone (3)	1.77	2.0
1T3 backbone (4)	1.42	1.9

2. Mini prep of DH5a with kit plate 1G (red colony)

Ø 5 tubes are prepared labeled as 1G (1) to (5)

Ø Store at -20oC

Ø Nanodrop

	ng/ul
1G (1)	6.37
1G (2)	0.04
1G (3)	0.22
1G (4)	4.48
1G (5)	0.04

3. Run gel (3-way 1T3)

Ø Show banding at ~4000-5000

4. Gel recovery of 3-way 1T3

5. Double digestion of 3-way 1T3

6. Run gel

Ø No banding shown

7. Restriction cut of 1G (red colony)

8. Run gel 1G (red colony)

Ø Band at ~1000 and ~3000-4000

9. Transformation

Plasmid: T71K3 and HR1T3

DH5a competent cells prepared on 30/6

10. Spread plates

11. Prepared 5 conical flasks with the following components

Ø LB with 50μl IPTG + 100 μl Antibiotic K

Ø LB of 0.4 M NaCl with 50 μl IPTG + 100 μl Antibiotic K

Ø LB of 0.6 M NaCl with 50 μl IPTG + 100 μl Antibiotic K

Ø LB of 0.4 M KCl with 50 μl IPTG + 100 μl Antibiotic K

Ø LB of 0.6 M KCl with 50 μl IPTG + 100 μl Antibiotic K

12. Transformed 5 ml DH5α with pET27b start culture and put them into the incubator at 37°C in the common room.

19 July (Tue)

1. Pick colonies that we spread yesterday

Ø T71K3 (with antibiotics K added) x2

Ø HR1T3 x2

2. Inoculation at 37oC, 250rpm for 6hrs

Ø Failed

3. Add 500 μl DH5α start culture into 5 respective conical flasks prepared on 18/7

Ø record the OD600 of 5 conical flasks and the results are recorded.

Time (h)	LB+IPTG	0.4M NaCl +LB +IPTG	0.6M NaCl +LB +IPTG	0.4M KCl+ LB+ IPTG	0.6M KCl + LB +IPTG
0	0.008	0.013	0.013	0.011	0.004
0.5	0.023	0.021	0.021	0.027	0.017
1.0	0.025	0.013	0.015	0.009	0.005
1.5	0.049	0.026	0.010	0.012	0.005
2.0	0.083	0.017	0.011	0.013	0.003
2.5	0.209	0.033	0.015	0.045	0.010
3.0	0.382	0.107	0.058	0.107	0.054
3.5	0.567	0.234	0.082	0.177	0.062
4.0	0.783	0.329	0.126	0.288	0.128
4.5	0.973	0.485	0.165	0.432	0.206
5.0	1.214	0.673	0.258	0.662	0.252
5.5	1.454	0.906	0.386	0.845	0.336
6.0	1.554	1.132	0.450	1.078	0.457

4. Transformed 3 start cultures of DH5α with pET27b with the total volume of 5ml with the following components

Ø 5ml LB + 5 μl K

Ø 3ml LB + 2ml of 1M NaCl + 5 μl K (LB with 0.4M of NaCl)

Ø 2ml LB + 3ml of 1M NaCl + 5 μl K (LB with 0.6M of NaCl)

20 July (Wed)

1. Pick colony

Ø HR1T3 X1

Ø T71K3 X1

Ø Red colony X3

Ø All with antibiotics added

2. Incubation

Ø 37^oC, 250rpm, 6hours

3. Transfer

Ø 37^oC, 240rpm, overnight

4. Prepared the autoclaved the 4 solutions

Ø 500 ml LB X2

Ø 500 ml LB constituting 1M NaCl X1

Ø 500 ml LB constituting 1M KCl X1

5. Prepared 5 conical with the following constitute

Ø LB with 50μl IPTG

Ø LB of 0.4 M NaCl with 50 μl IPTG

Ø LB of 0.6 M NaCl with 50 μl IPTG

Ø LB of 0.4 M KCl with 50 μl IPTG

Ø LB of 0.6 M KCl with 50 μl IPTG

6. Transformed 5 DH5α with pET27b start solution with different NaCl and KCl concentrations

Ø 5ml LB + 5 μl antibiotic K

Ø 5ml of 0.4M NaCl + 5 μl antibiotic K

Ø 5ml of 0.6M NaCl + 5 μl antibiotic K

Ø 5ml of 0.4M KCl + 5 μl antibiotic K

Ø 5ml of 0.6M KCl + 5 μl antibiotic K

21July (Thu)

1. Midi Prep of red colony (1G) & HR

Ø 3 samples of 1G: 35.7ng/ul, 27ng/ul, 124ng/ul

Ø 1 sample of HR: 5.0ng/ul

2. Digestion of 1G red colony using 1G(1) & 1G(3)

Ø 1G(1)

Buffer 3	2.5ul
100X BSA	0.25ul
Eco R1	0.5ul
Pst 1	0.5ul
DNA	14ul (500ng)
H20	7.25ul

Ø 1G(3)

Buffer 3	2.5ul
100X BSA	0.25ul
Eco R1	0.5ul
Pst 1	0.5ul
DNA	4ul (500ng)
H20	17.25ul

Ø Incubated at 37^oC, 2hours

3. Run gel using digestion products

Ø Band size: ~1000 and 2000-2500kb

4. Could not measure the absorbance of LB of 0.6M NaCl, 0.4M KCl and 0.6M KCl because the start culture of DH5α cannot grow properly. The absorbance of start culture of LB and LB of 0.4M NaCl prepared on 20/7/2011 without adding DH5α were measured. Other than that, we also measured the absorbance of LB+ IPTG and LB of 0.4M NaCl+ LB + IPTG

Ø Result

	LB + IPTG	LB of 0.4M of NaCl
Start culture absorbance (1ml)	1.323	0.464

5.

Time (h)	LB+ IPTG	LB of 0.4M NaCl + IPTG
0	0.021	0.014
0.5	0.026	0.024
1.0	0.045	0.033
1.5	0.043	0.036
2.0	0.133	0.064
2.5	0.245	0.105
3.0	0.359	0.159
3.5	0.555	0.228
4.0	0.720	0.303
4.5	0.927	0.466
5.0	0628 (2X)	0.618
5.5	0.846(2X)	0.815
6.0	0.899(2X)	0.610(2X)

22July (Fri)

1. Run gel (HR)

2. Transformation

Ø 3A pSB1C3

Ø 5A pSB1K3

Ø 7A pSB1T3

Ø T7 1K3

Ø DH5a competent cells

3. Spread plate

Ø 3C plates of 3A-pSB1C3

Ø 4K plates of 5A-pSB1K3

Ø 4T plates of 7A-pSB1T3

Ø 3K plates of T7-1K3

23July (Sat)

1. Digestion using 1G(3) prepared on 21July2011

Ø incubated at 37^oC, 2hours

Ø product stored in 4^oC fridge with label 1G backbone 23h

2. Pick colony & Inoculation

Ø using plates prepared on 22July

- 3A pSB1C3

- 5A pSB1K3

- 7A pSB1T3

- T7-1K3

Ø 3 snap caps containing 5ml LB and corresponding antibiotics are prepared for each type of plate.

- 3A pSB1C3 23/7 莊

- 5A pSB1K3 23/7 莊

- 7A pSB1T3 23/7 莊

- T7-1K3 23/7 莊

- Snap caps are put in common shaker

24July (Sun)

1. Mini prep using cultures prepared yesterday, 24 samples are made:

Ø Stored in -20^oC fridge

- 3A1C3 (1-8)

- 7A1T3 (1-4)

- 5A1K3 (1-4)

- T71K3 (1-8)

Week 8 Monday 25 July – Sunday 31 July

25 July (Mon)

1.      Restriction cut

Ø   1G (1A3)

2.      Run gel to extract backbone 1A3

Ø   2 bands observed: ~1000 and ~2000-3000

3.      Gel recovery

4.      Nanodrop of 24 samples made on 24/7

ng/ul

ng/ul

ng/ul

3A1C3 (1)

58.7

5A1K3 (1)

48.0

T71K3 (1)

47.6

3A1C3 (2)

42.8

5A1K3 (2)

24.2

T71K3 (2)

46.8

3A1C3 (3)

51.8

5A1K3 (3)

12.1

T71K3 (3)

47.6

3A1C3 (4)

58.1

5A1K3 (4)

12.0

T71K3 (4)

33.9

3A1C3 (5)

29.0

7A1T3 (1)

35.2

T71K3 (5)

47.1

3A1C3 (6)

28.6

7A1T3 (2)

24.2

T71K3 (6)

43.6

3A1C3 (7)

13.9

7A1T3 (3)

27.6

T71K3 (7)

41.4

3A1C3 (8)

34.9

7A1T3 (4)

27.1

T71K3 (8)

31.9

5.      Transformed competent DH5α cells with vector pET27b HR and then spread them on K plate. The plate was then put into the incubator at 37°C

26 July (Tue)

1.      Restriction cut using HR and T7

Buffer 3

2.5ul

BSA

0.25ul

EcoR1

0.5ul

Pst1

0.5ul

DNA (35.3ng/ul)

14.25ul

H₂O

7.0ul

Total

25ul

Buffer 3

2.5ul

BSA

0.25ul

EcoR1

0.5ul

Pst1

0.5ul

DNA (180ng/ul)

21.25ul

Total

25ul

Ø   Incubate at 37oC for 2hrs

2.      Run gel

Ø   Result:     HR: ~800bp

                          T7: 200bp

3.      Gel recovery

4.      3-way assembly

H2O

11ul

10X T4 DNA ligase buffer

2ul

T4 DNA ligase

1ul

HR

2ul

T7

2ul

1A3

2ul

Total

20ul

5.      Prepared 5 conical flasks

Ø   LB with 50μl IPTG

Ø   LB of 0.4 M NaCl with 50 μl IPTG

Ø   LB of 0.6 M NaCl with 50 μl IPTG

Ø   LB of 0.4 M KCl with 50 μl IPTG

Ø   LB of 0.6 M KCl with 50 μl IPTG

6.      5 start cultures were also prepared

Ø   5ml LB + 5 μl antibiotic K

Ø   5ml of 0.4M NaCl + 5 μl antibiotic K

Ø   5ml of 0.6M NaCl + 5 μl antibiotic K

Ø   5ml of 0.4M KCl + 5 μl antibiotic K

Ø   5ml of 0.6M KCl + 5 μl antibiotic K

27 July (Wed)

1.      Transform terminator (iGEM plate2 24C) into DH5a

2.      Restriction cut

Ø   HR1K3 and HR-T71A3 (3-way assembly) by EcoR1 and Pst1

3.      Run gel

Ø   Ladder mix together, cannot see size

4.      Spread plates

Ø   6 K-plates of 24C terminator

5.      Measured the absorbance of the 5 conical flasks prepared on the 26/7 by cell counting machine with the following result

Start culture

LB

KCl 0.4M

NaCl 0.4M

KCl 0.6M

NaCl 0.6M

First measurement (4X)

0.430

0.674

0.410

0.380

Second measurement

(4X)

0.401

0.636

0.431

0.488

0.348

Ø   Preciously, we use the qViro cell counter

Result

Time (h)/ culture

LB

KCl 0.4M

NaCl 0.4M

KCl 0.6M

NaCl 0.6M

0

0.023

0.046

0.027

0.022

0.025

0.5

0.025

0.038

0.029

0.029

0.027

1.0

0.036

0.032

0.026

0.039

0.048

1.5

0.078

0.038

0.035

0.043

0.041

2.0

0.142

0.104

0.057

0.038

0.040

2.5

0.295

0.154

0.113

0.053

0.053

3.0

0.437

0.287

0.179

0.077

0.067

3.5

0.601

0.408

0.270

0.126

0.104

4.0

0.796

0.580

0.373

0.162

0.146

28 July (Thur)

1.      Collect plates from 27/7, store at 4^oC

2.      Run gel

Ø   3-way assemblt plasmid made yesterday

Ø   No bands shown

3.      Restriction cut of pSB1A3

Buffer 2

2.5ul

BSA

0.25ul

EcoR1

0.5ul

Pst1

0.5ul

H₂O

16ul

DNA

5.25ul

Total

25ul

Ø   37oC for 2hrs

4.      Run gel

Ø   No bands shown

5.      Measured the absorbance change over time of the flask LB IPTG (100ml) with DH5α transformed with pET27b measured and counted the cells in CSLB G12

Result

Time (h)

OD₆₀₀

Cell Count (particle per ml)

0

-0.004

4.5 X 10⁸

0.5

-0.002

5.2 X 10⁸

1.0

±0.000

6.7 X 10⁸

1.5

+0.002

8.7 X 10⁹

2.0

+0.007

1.8 X 10⁹

2.5

+0.026

1.8 X 10⁹

3.0

+0.041

3.1 X 10⁹

3.5

+0.061

4.8 X 10⁹

29July (Fri)

1.      Run gel

Ø   45min, 120V

Ø   6X loading dye: 4ul, DNA: 20ul

Lane

1

2

3

4

5

6

7

8

ladder

1G cut

T7 cut

HR cut

Ø   Result
- lane 3: 1000-1500bp & 2000-3000bp
- lane 4: 100-200bp
- lane 5: 500-1000bp

2.      Gel clean of T7, HR, 1G

Ø   Labeled: “T7 28/7 Mo”, “HR 28/7 Mo”, “1G 29/7 Mo”

Ø   Stored in 4^oC fridge

3.      Ligation using T7, HR, 1G

Ø   Incubated at 16^oC

HR

7ul

T7

7ul

1G

2ul

Ligation buffer

2ul

T4 ligase

1ul

H2O

1ul

TOTAL

20ul

4.      100mM MQAE solution and alinquoted into 1.5ml black microcentrifuge tube with 500μl each stored at -20°C. 5μl MQAE and 95μl salt solution were added to microplate and microplatereaders was used in SC 127.

Ø   MQAE

Mass (mg)

100

Molar mass (g mol^-1 )

326.1893

Concentration (mM)

100

Volume made (ml)

3.066

Ø   Concentration of the salt solution with 2-fold dilution

                            i.                100mM NaCl

                          ii.                50mM NaCl

                        iii.                25mM NaCl

                         iv.                12.5mM NaCl

                           v.                6.25mM NaCl

                         vi.                3.125mM NaCl

Ø   Final concentration of MQAE in microplate reader is 5mM

30July (Sat)

1.      PCR amplification of T7 & HR

Ø   Labeled as T7 1, T7 2, HR

31July (Sun)

1.      Run gel using PCR product

2.      Gel recovery

Ø   1 tube of HR, 1 tube of T7, each of 30ul

3.      Digestion

Ø   1G, 3A1C3, 5A1K3, 7A1T3

Ø   Use products to run gel, obtain backbone

4.      Ligation

Ø   (HR, T7) & (1C3, 1K3, 1T3 backbone)

Ø   4 samples: T71K3, T71T3, HR1K3, HR1C3

Ø   Incubated at 16^oC, overnight

5.      Transformation

Ø   use competent cell prepared on 30/6

Ø   4 samples: 3A1C3, 3A1T3, 5A1K3, 1G

Ø   Spread plates: using A,K,C,T plates, each type 2 plates

Week 9 Monday 1 Aug – Sunday 7 Aug

1 Aug (Mon)

1. PCR amplification of T7 and HR

2. PCR purification of T7 and HR

3. Transformation

Ø 1G, 3A, 5A, 7A

-Used DH5a competent cells

-2 sets are prepared

Ø HR1C3, HR1K3, T71T3, T71K3

-Used DH5a competent cells

-2 sets are prepared

Ø chloride sensor

-Used DH5a competent cells

-1 tube is prepared

4. Spread plate

Ø One plate is used for each tube of transformed competent cells

Ø All plates stored in incubator at 37^oC

5. Run gel to check size

Ø Result: HR: ~800

T7: ~100-200

6. Prepared 1 flask comprising 100ml LB, 50μl IPTG and 100μl antibiotic K and also 1 DH5α with pET27b HR start culture

2 Aug (Tue)

1. Inoculation

Ø HR1K3 (failed)

Ø HR1C3

Ø T71K3 (failed)

Ø T71T3

Ø 3A

Ø 5A

Ø 7A

Ø 1G

Ø Chloride sensor (failed)

2. Restriction cut

Ø HR, T7, 1T3

3. Ligation

Ø HR1T3 and T71T3

Ø Store at -16^oC overnight

4. Transfer to 125ml conical flask for MIDI Pre

5. Transformation

Ø 24C terminator and DH5a used

Ø each spread on 2 plates

6. Prepare agar plate

Ø C plates x18

Ø A plates x20

Ø K plates x21

7. Absorbance of the start culture was measured and the cells were counted

Time (h)	OD₆₀₀	Cell count (cell/ml)
0	0.000	4 X 10⁸
0.5	0.003	1.4 X 10¹⁰
1.0	0.008	8.6 X 10⁷
1.5	0.022	3.3 X 10⁸
2.0	0.142	3 X 10⁸
2.5	0.203	7 X 10⁸
3.0	0.263	/

Result

Ø Brian suggested that the abnormality of the cell count data might be due to the growing size of the bacteria. At 3.0 hour, signal could not be obtained as well as the reason.

3 Aug (Wed)

1. Mini prep

Ø 3A

Ø 5A

Ø 7A

Ø T71T3

Ø Cl sensor

2. 3-way assembly

	Ng/ul	260/280
3-way	12.8	1.93
Cl sensor	1.2	1.85
T71T3	13.0	1.93
HR1C3	103.0 (15.9)	1.33
5A	35.3	1.87
7A	24.2	1.90

3. Concentration of the NaCl solution was prepared as follow by serial dilution

Ø 1000mM NaCl

Ø 500mM NaCl

Ø 100mM NaCl

Ø 50mM NaCl

Ø 25mM NaCl

Ø 12.5mM NaCl

Ø 6.25mM NaCl

Ø 3.125mM NaCl

4. Concentration of MQAE was also prepared as follow by serial dilution

Ø 100mM MQAE

Ø 10mM MQAE

Ø 1mM MQAE

5. 95μl of NaCl solutions and 5 μl of MQAE solutions were added into the microplate in the position as shown below.

As a result, the final MQAE concentration

Ø A1-A4,B1-B4, C1-C4, D1-D4, E1-E4, F1-F4, H1-H4 are 5mM

Ø A5-A8, B5-B8, C5-C8, D5-D8, E5-E8, F5-F8, H5-H8 are 0.5mM

Ø A9-A12, B9-B12, C9-C12, D9-D12, E9-E12, F9-F12, H9-H12 are 0.05mM

Ø The plate with aluminum foil was wrapped and put in the incubator at 37°C for 1 hour and measured emission by microplate reader

Ø Result

1	2	3	4	5	6	7	8	9	10	11	12
5480	5633	5428	5554	3013	3267	3058	3209	1249	1335	1341	1267
8990	9199	9483	9446	5235	5222	5241	4649	1545	1646	1634	1630
763	21493	21870	23048	11411	11549	11711	10767	2564	2513	2549	2413
25542	29517	27811	29312	14561	14986	14392	14646	3056	3018	2848	2839
30141	32470	33457	27985	17182	17219	14902	19335	3351	3318	2974	2931
37055	36110	39855	35193	18985	18717	18398	16975	3565	3349	3216	3366
34258	36477	37255	35236	18320	18588	18778	6182	3425	3211	3424	3265
36893	37123	37393	35904	13574	25312	19447	1334	3386	3529	3191	3364

A: 1000mM NaCl

B: 500mM NaCl

C: 100mM NaCl

D: 50mM NaCl

E: 25mM NaCl

F: 12.5mM NaCl

G: 6.25mM NaCl

H: 3.125mM NaCl

4 Aug (Thu)

1. Mini prep

	ng/ul	260/280
24C (1)	44.1	1.77
24C (2)	59.7	1.76
T71K3 (1)	---	---
T71K3 (2)	41.7	1.87
HR1K3 (1)	18.5	1.92
HR1K3 (2)	29.5	1.78
3A (1)	---	---
3A (2)	50.7	1.88
5A (1)	44.4	1.85
5A (2)	---	---
1G (1)	142.9	1.86
1G (2)	52.4	1.89
7A	43.5	1.86

Ø 3A (1) is red while 3A (2) is milky

Ø 1G (1) is red while 1G (2) is milky

2. Double digestion

Ø T71K3

Buffer 2	2ul
BSA	0.2ul
EcoR1	0.5ul
Spe1	0.5ul
T71K3 (prepared on 4/8)	16.8ul
Total	20ul

Ø HR1C3

Buffer 2	2ul
BSA	0.2ul
EcoR1	0.5ul
Pst 1	0.5ul
HR1C3 (prepared on 3/8)	16.8ul
Total	20ul

Ø 7A

Buffer 2	2ul
BSA	0.2ul
EcoR1	0.5ul
Pst 1	0.5ul
7A (prepared on 3/8)	4ul
H2O	12.8ul
Total	20ul

-37^oC, 2hours

3. 3-way ligation

T4 ligase buffer	2ul
T4 ligase	1ul
T7	10ul
HR	4ul
Vector (7A)	3ul
Total	20ul

4. Inoculation

Ø Pick colony of white from T71T3, HR1T3

5 Aug (Fri)

1. Nanodrop

Samples prepared on 4/8	ng/ul	260/280
HR1T3 (1)	10.3	1.98
HR1T3 (2)	12.2	1.91
T71T3 (1)	10.5	1.82
T71T3 (2)	12.4	1.86

2. Transformation

Ø used DH5a competent cells

Ø only 1 set is prepared using 3-way ligation products

Ø Plate was spreaded after transformation using T plates

(plates labeled: 5/8transformation, 3A ligation)

Ø store st 37oC

3. Pick colonies

Ø pick from T71T3

Ø 3 colonies are picked

Ø inoculate for 12-16hrs

Ø 3 snapcap tubes, 37^oC, 250rpm

7 Aug (Sun)

1. inoculation form plates HR-T7-1T3 prepared on 6/8

2. pick 3 colonies to snap-cap tubes with 6ml LB, then shake at 250rpm, 37^oC for 12-16 hrs

3. Prepared and inoculated four tubes of the start culture with the volume of 10ml , which are 0.4M NaCl LB with light, 0.4M NaCl LB without light , LB with light, and LB without light. But we didn’t add IPTG.

4. Measured OD 600 of four start culture

	0.4M NaCl LB with light	0.4M NaCl LB without light	LB with light	LB without light
OD600 (2X)	0.230	0.401	0.388	0.690

5. Spin four tubes of start culture separately to remove the LB/NaCl LB then store in -80°C refrigerator.

6. Prepared four tubes of start culture with the total volume of 10 ml, which 0.4M NaCl LB with light, 0.4M NaCl LB without light, LB with light and LB without light and inoculated them overnight 5μl of 1M IPTG was added

Week 10 Monday 8 Aug – Sunday 14 Aug

8 Aug (Mon)

1.      Mini prep 3-way ligation products prepared on 7/8

Result

ng/ul

260/280

3-way ligation

11.5

1.83

HR-T7-1T3 (1)

13.9

1.86

HR-T7-1T3 (2)

13.2

1.91

T71T3 (1), prepared on 6/8

29.4

1.52

T71T3 (2) , prepared on 6/8

7.3

1.69

T71T3 (3) , prepared on 6/8

5.4

1.74

Ø   DNA ladder used is from Fermentas GeneRuler 1kb DNA, #SM1331

2.      Run gel

3.      Double digestion

Buffer 2

2ul

BSA

0.2ul

EcoR1

0.5ul

Spe 1

0.5ul

T71T3 (prepared on 6/8)

16.8ul

Total

20ul

Buffer 2

2ul

BSA

0.2ul

XBa 1

0.5ul

Pst 1

0.5ul

HR1K3 (prepared on 4/8)

16.8ul

Total

20ul

Buffer 2

2ul

BSA

0.2ul

EcoR1

0.5ul

Pst 1

0.5ul

3A (prepared on 4/8)

2ul

H2O

14.8ul

Total

20ul

Ø   37^oC, 2hrs

4.      Ligation

T4 ligase buffer

2ul

T4 ligase

1ul

T7

7ul

HR

7ul

Vector pSB1C3 from 3-way

3ul

Total

20ul

Ø   16^oC overnight

5.      Measured OD₆₀₀of the four start cultures of 10ml of IPTG made on 7/8 then spin four tubes of start culture separately to remove the LB/NaCl LB

Ø   Put in the -80°C refrigerator for storage.

6.      Prepared again four tubes of 10ml of start culture

Ø   LB of 0.4M NaCl with light

Ø   LB of 0.4M NaCl without light

Ø   LB with light

Ø   LB without light

Ø   Inoculated them with the addition of 5μl of 1M IPTG.

7.          Measured OD₆₀₀ of the four start cultures.

Result:

OD₆₀₀of the four start cultures of 10ml of IPTG made on 7-8-2011

LB of 0.4M NaClwith light

LB of 0.4M NaClwithout light

LB with light

LB without light

OD₆₀₀(2X)

0.328

0.503

0.875

0.934

OD₆₀₀ of the four start cultures were measured.

LB of 0.4M NaClwith light

LB of 0.4M NaClwithout light

LB with light

LB without light

OD₆₀₀ (2X)

0.357

0.526

0.390

0.627

Ø   Spin four tubes to remove the LB/LB NaCl

Ø   Store in -80°C refrigerator.

9 Aug (Tue)

1.      Transformation of 3-way ligation clone prepared on 8/8

2.      Transformation of T71T3 mini prep product prepared on 6/8

3.      Transformation of 3-way ligation prepared on 8/8

Ø   All experiment were done following the standard transformation protocol, except that for: 1. 2ul added, 2. 2ul added, 3. 5ul added

4.      Spread plates with antibiotics C, T respectively

5.      Pick colony from T71T3, HR1T3, 3-way ligation plates from refrigerator and then inoculate overnight

6.      We unfortunately make a mistake that because the OD was measured and spinned all of the solution, whereas the start solution should be spinned with accordance with the volume calculated from the measurement of the OD. Therefore, the whole experiment needs to be redone.

10 Aug (Wed)

1.      Mini prep of T71T3, HR1T3, 3-way ligation from 9/8

ng/ul

260/280

T71T3 (1)

111.0

1.84

T71T3 (2)

109.9

1.85

T71T3 (3)

147.9

1.90

HR1T3 (1)

103.0

1.89

HR1T3 (2)

98.9

1.85

HR1T3 (3)

107.6

1.84

3A (1)

101.2

.182

3A (2)

123.6

1.88

3A (3)

210.0

1.87

Ø   all are using 50ul H2O to elute al the final step, except that all tubes of 3 had been added 30ul to increase concentration

2.      inoculation

Pick 3 colonies from 3 plates respectively, totally of 9, from

Ø  3-way ligation(8/8) after mini prep done on 9/8

Ø  3-way ligation(8/8) done on 9/8

Ø  T71T3 (6/8) done on 9/8

Labeled as follows:

3-way post miniprep (1)

3-way post miniprep (2)

3-way post miniprep (3)

3-way (1)

3-way (2)

3-way (3)

T71T3 (1)

T71T3 (2)

T71T3 (3)

3.      Inoculated 4 DH5α with pET27b HR start cultures namely,

Ø   LB + IPTG +K with light

Ø   LB + IPTG +K without light wrapped with aluminum foil

Ø   LB + 0.4M NaCl + IPTG +K with light

Ø   LB  + 0.4M NaCl + IPTG +K without light wrapped with aluminum foil

11 Aug (Thu)

1.        Miniprep of 9 tubes prepared on 10/8

Tube

Concentration (ug/ul)

260/280

3A Post mini prep (1)

96.1

1.91

3A Post mini prep (2)

41.6

1.96

3A Post mini prep (3)

100.5

1.85

3A (1)

104.7

1.85

3A (2)

68.8

1.87

3A (3)

74.7

1.96

T71T3 (1)

87.3

1.79

T71T3 (2)

115.8

1.85

T71T3 (3)

135.9

1.91

Ø   all no. 3 tubes are eluted with 30ul 60^oC water to increase yield and concentration

2.        Transform plate 2 24C terminator

Ø   use competent cell made on 30/6

Ø   24C terminator mini-prep on 4/8 by Eric

Ø   Plate stored at 37^oC incubator, overnight

Ø   Remaining transformed 24C terminator stored at -20^oC,
labeled as “ transf. 24C, 11/8,ii)

3.        Sequencing

Ø   9 tubes prepared on 10/8, 9 tubes prepared on 11/8

4.      Measured the OD₆₀₀ of the 4 start cultures prepared on 10/8

LB of 0.4M NaClwith light

LB of 0.4M NaClwithout light

LB with light

LB without light

OD₆₀₀ (2X)

0.266

0.267

0.302

0.286

5.      10% SDS was diluted into 2% SDS by adding the autoclaved 40ml of distilled water and 10ml of 10% SDS based on the insensitivity of SDS

6.      Measure the OD600 of the second set without IPTG

LB of 0.4M NaClwith light

LB of 0.4M NaClwithout light

LB with light

LB without light

OD₆₀₀ (2X)

0.094

0.096

0.350

0.355

Volume to bespinned (ml)

*All

*All

4.5

4.5

All volume to be spinned because the OD of NaCl sets are too small so the total volumes are still inadequate. As a result, a back up set without IPTG by inoculation was prepared and collected on 12-8-2011. It was found that the DH5α plate has been used up for cline picking so later transformation and plate spreading is need. Besides, it was found that there is only little amount of 1000K anti-biotic too.

12 Aug (Fri)

1.        Inoculation

Ø   pick 3 colonies from plate “24C terminator transformed 11Aug”

2.        Measured the OD600 of the back up set of bacteria.

LB of 0.4M NaClwith light

LB of 0.4M NaClwithout light

LB with light

LB without light

OD₆₀₀ (2X)

0.459

0.290

0.768

0.847

Volume to bespinned (ml)

3.5

5.5

2

1.9

3.        Pellets after spinning down were stored in -80°C refrigerator

Ø   2% of 100μl of SDS was used to lyse the cells in 99°C for 5 minutes

Ø   Serial dilution of the bacteria in 2X, 4X, 8X and 16X and of salt like previously done on 3/8 were done

Ø   The MQAE and the solutions were added into the microplate and incubated for an hour

4.        Measured the MQAE fluorescence with microplate reader

Ø   The data were abnormal and Frankie deduced that it may be due to the bubbles of 2% SDS

13 Aug (Sat)

1.      Miniprep of 24C terminator prepared on 12/8

Ø   3 tubes are labeled as “24C (1) 13/8”, “24C (2) 13/8”, “24C (3) 13/8”

Week 11 Monday 15 Aug – Sunday 21 Aug

15 Aug (Mon)

1. Transformed pET27bHR into DH5α and spread on a K plate

16 Aug (Tue)

1. Inoculated LB IPTF, 0.4M NaCl IPTG, 0.4M NaCl IPTG with light, 0.4M KCL IPTG and 0.4M KCl IPTG without light with DH5α pET27b

17 Aug (Wed)

1. Measured the OD 600 of the back up set of the bacteria

set up	LB + IPTG without light	NaCl + IPTG with light	NaCl + IPTG without light	KCl + IPTG with light	KCl + IPTG without light
OD₆₀₀ (2X)	0.857	0.724	0.733	0.316	0.660
Volume to bespinned down (ml)	1.762	2.086	2.060	4.778	2.288

2. 2% of SDS was spinned to lyse the cells in 99°C for 5 minutes with 100μl each, Serial dilution of the bacteria in 8X, 16X, 32X, 64X and of salt like previously done was carried out. The MQAE and solutions were added into the microplate and inoculated for an hour.

3. Measured the MQAE fluorescence with microplate reader

Ø The data was also abnormal and Frankie deduced that it may be also due to the bubbles of the 2% SDS.

4. Inoculated the following tubes with DH5α pET27b

Ø LB + IPTG

Ø 0.4M NaCl + IPTG without light

Ø 0.4M NaCl + IPTG with light

Ø 0.4M KCl + IPTG without light

Ø 0.4M KCL + IPTG with light

Ø ( 10μl K and 5μ of IPTG)

18 Aug (Thu)

1. Run gel with PCR products (T7, HR)

Ø Result: HR did not show band with 800 bp

2. Run gel using PCR product (T7)

Ø Result: the samples show unclear result

3. PCR with T7 & HR

4. PCR purification of PCR product of step 3

5. Measured the OD600 of the back up set of the bacteria

set up	LB + IPTG without light	NaCl + IPTG with light	NaCl + IPTG without light	KCl + IPTG with light	KCl + IPTG without light
OD₆₀₀(2X)	0.862	0.242	0.751	0.797	0.252
Volume to bespinned down (ml)	2.8	10	3.2	3.0	9.6

6. 2% of SDS was spin to lyses the cells in 99°C for 5 minutes with 100μl each, Serial dilution of the bacteria in 8X, 16X, 32X, 64X and of salt like previously done was carried out. The MQAE and solutions were added into the microplate and inoculated for an hour.

7. MQAE fluorescence was measured with microplate reader

Ø The data was abnormal and may be due to the bubbles of the 2% SDS.

19Aug (Fri)

1. Run gel to check size of PCR amplified T7 & HR prepared on 18/08

7. HR: result failed, at ~100bp

8. T7: result ok, at 100-200bp

20Aug (Sat)

1. Inoculation of 3 plate prepared last night

Ø 6 tubes prepared

Ø Abandoned because MidiPrep cannot be done on Sunday, will do miniprep instead)

2. PCR with HR templates (2tubes)

3. Restriction vut T7 prepared on 18/8 & backbone 5A1C3

Ø 1tube of T7

Buffer	2ul
BSA	0.2ul
EcoR1	0.4ul
Pst1	0.4ul
T7	15ul
H20	2ul
TOTAL	20ul

Ø 2tubes of backbone

Buffer	2ul
BSA	0.2ul
EcoR1	0.4ul
Pst1	0.4ul
backbone	10ul
H20	7ul
TOTAL	20ul

4. PCR purification of PCR product HR (2tubes) (continued step 2)

5. Run gel to check size of HR purified product

Ø Run 50ul of each HR and do gel recovery

Ø Each lane of HR with 25ul

Ø Result: size correct, ~800bp

Lane	1	2	3	4	5	6
	---	100bp ladder	HR-1	HR-1	HR-2	HR-2

6. Ligation of T7 & 1C3, overnight

Ø 3 tubes

T4 ligase buffer	2ul
T4 ligase	1ul
T7	3ul
1C3	14ul
TOTAL	20ul

7. Inoculation of 24C terminator DH5a

21 Aug (Sun)

1. Miniprep 6 tubes of 24C terminator

2. Transformation:

Ø T71C3 (1) prepared on 20/8

Ø 1G1A3

Ø 3A1C3

Ø 5A1K3

Ø 7A1T3

Ø Spread plate:
- white colonies: T71C3
- red colonies: 1G1A3, 3A1C3, 5A1K3, 7A1T3

3. Restriction cut of HR and backbone 1C3

Ø HR: 10ul

Ø Backbone X2: total 40ul

4. Ligation of HR & 1C3, overnight

Ø 3 tubes labeled as “HR1C3(1-3)”

5. Inoculated with DH5α pET27b

Ø LB + IPTG

Ø 0.4M NaCl + IPTG without light

Ø 0.4M NaCl + IPTG with light

Ø 0.4M KCl + IPTG without light

Ø 0.4M KCL + IPTG with light

Ø ( 10μl K and 5μ of IPTG)

Week 12 Monday 22 Aug – Sunday 28 Aug

22 Aug (Mon)

1.      Inoculation

Ø   37^oC, 240rpm

Ø   1G1A3, 7A1T3 àwhite colonies

Ø   3A1C3, 5A1K3àred colonies

Ø   Did not pick T71C3

2.      Nanodrop of 24C (X6) & HR (X3)

ug/ul

260/280

24C (1)

10.5

1.72

24C (2)

7.9

1.78

24C (3)

8.2

1.73

24C (4)

7.9

1.59

24C (5)

11.7

1.54

24C (6)

26.7

1.37

HR (1)

6

1.67

HR (2)

4.9

1.80

HR (3)

8

1.07

3.      Transformation

4.      Transfer of 1G1A3, 7A1T3, 3A1C3, 5A1K3

Ø   each type 2 tubes

5.      inoculation of transferred product

6.      Measure OD600 of the back up set of the bacteria

Set up

LB + IPTG without light

NaCl + IPTG with light

NaCl + IPTG without light

KCl + IPTG with light

KCl + IPTG without light

OD₆₀₀(2X)

0.303

0.350

0.637

0.689

0.269

Volume to bespinned down (ml)

8

6.9

3.8

3.5

9

7.      2% of SDS was spin to lyses the cells in 99°C for 5 minutes with 400μl each

Ø   Serial dilution of the bacteria in 8X, 16X, 32X, 64X and of salt like previously done was carried out

Ø   95μl of each concentration was added into each well

Ø   5μl of MQAE was added into each well afterwards

Ø   A duplicate was made. Sample prepared by Frankie and 5 μl of MQAE were added with the same volume

8.      Measured the MQAE fluorescence with microplate reader

Ø   Result of 0.4M NaCl IPTG without light lay in the range of our standards

9.      Inoculated the following tubes with DH5α pET27b

Ø   LB + IPTG

Ø   0.4M NaCl + IPTG without light

Ø   0.4M NaCl + IPTG with light

Ø   0.4M KCl + IPTG without light

Ø   0.4M KCL + IPTG with light

Ø   ( 10μl  K and 5μ of IPTG)

23 Aug (Tue)

1.      midiprep 3A1C3, 5A1K3

2.      transform 24C, chloride sensor, 1G1A3, 7A1T3,T71C3 with DH5a cells

Ø   spread the above to agar plates

Ø   each 2 plates

3.      pick colonies and inoculation of the above

Ø   result: 1G1A3 & chloride sensor are too dense, others are ok

4.      Measured the OD600 of the bacteria

Set up

LB + IPTG without light

NaCl + IPTG with light

NaCl + IPTG without light

KCl + IPTG with light

KCl + IPTG without light

OD₆₀₀(2X)

0.923

0.875

0.318

0.396

0.706

Volume to bespinned down (ml)

2.62

2.77

7.6

6.11

3.43

5.      2% of SDS was spinned to lyse the cells in 99°C for 5 minutes with 400μl each

Ø   Serial dilution of the bacteria in 8X, 16X, 32X, 64X and of salt like previously done was carried out

Ø   95μl of each concentration was added into each well

Ø   A duplicate was made. Sample prepared by Frankie and 5 μl of MQAE were added with the same volume.

Ø   MQAE fluorescence was measured by microplate reader and the results were sent to Frankie.

6.      Routinely, the following tubes are inoculated with DH5α pET27b

Ø   LB + IPTG

Ø   0.4M NaCl + IPTG without light

Ø   0.4M NaCl + IPTG with light

Ø   0.4M KCl + IPTG without light

Ø   0.4M KCL + IPTG with light

Ø   ( 10μl  K and 5μ of IPTG)

24 Aug (Wed)

1.      miniprep HR1C3

Ø   Result:

sample

Conc. (ng/ul)

260/280

HR1C3 (1)

30.5

1.74

(2)

24.7

1.81

(3)

19.9

1.94

(4)

22.1

1.97

(5)

28.3

1.80

(6)

50.0, 34.8, 21.0, 24.8, 25.3

1.94, 1.77, 2.25, 1.97, 1.91

(7)

42.1, 29.5

1.91, 1.92

(8)

24.6

1.78

(9)

31.4, 25.8

1.80, 1.91

(10)

18.9

1.91

(11)

19.2

1.99

(12)

48.2, 43.1, 45.8

1.88, 1.99, 1.92

2.      transform 1ul chloride sensor to 20ul DH5a competent cells

3.      Measured the OD600 of the bacteria

Set up

LB + IPTG without light

NaCl + IPTG with light

NaCl + IPTG without light

KCl + IPTG with light

KCl + IPTG without light

OD₆₀₀(2X)

0.667

0.148

0.107

0.178

0.181

Volume to bespinned down (ml)

1.81

9.5

9.5

9.5

9.5

4.      Then we use 2% of SDS to spin to lyse the cells in 99°C for 5 minutes with 400μl each

Ø   Serial dilution of the bacteria of 2-fold, 4-fold, 8-fold and 16-fold was carried out

Ø   The sample (A-I) prepared by Frankie was added into the well

Ø   95μl each and a duplicate was done

Ø   Finally, 5μl of MQAE was added into the wells with the samples

Ø   The microplate was put into the incubator at 37°C for 1 hour.

5.      Measured the MQAE fluorescence by microplate reader.

6.      Following tubes are inoculated with DH5α pET27b

Ø   LB + IPTG

Ø   0.4M NaCl + IPTG without light

Ø   0.4M NaCl + IPTG with light

Ø   0.4M KCl + IPTG without light

Ø   0.4M KCL + IPTG with light

Ø   ( 10μl  K and 12.5μ of 0.4M of IPTG)

25 Aug (Thu)

1.          Measured the OD₆₀₀ of the bacteria

Set up

LB + IPTG without light

NaCl + IPTG with light

NaCl + IPTG without light

KCl + IPTG with light

KCl + IPTG without light

OD₆₀₀(2X)

0.851

0.718

0.767

0.322

0.717

Volume to bespinned down (ml)

3.4

4.0

3.8

9.0

4.0

2.          Use 2% of SDS to spin to lyse the cells in 99°C for 5 minutes with 400μl each

Ø   Serial dilution of the bacteria of 2-fold, 4-fold, 8-fold and 16-fold was carried out

Ø   The sample (A-I) prepared by Frankie was added into the wells

Ø   95μl each and a duplicate was done

Ø   Finally, 5μl of MQAE was added into the wells with the samples

Ø   The microplate was put into the incubator at 37°C for 1 hour.

3.          Measured the MQAE fluorescence by microplate reader.

4.          Following tubes are inoculated with DH5α pET27b

Ø   LB + IPTG

Ø   0.4M NaCl + IPTG without light

Ø   0.4M NaCl + IPTG with light

Ø   0.4M KCl + IPTG without light

Ø   0.4M KCL + IPTG with light

Ø   ( 10μl  K and 12.5μ of 0.4M of IPTG)

26 Aug (Fri)

1.          Measured the OD₆₀₀ of the bacteria

Set up

LB + IPTG without light

NaCl + IPTG with light

NaCl + IPTG without light

KCl + IPTG with light

KCl + IPTG without light

OD₆₀₀(2X)

0.878

0.326

0.447

0.262

0.625

Volume to bespinned down (ml)

2.7

7.2

5.3

9.0

3.8

2.          Use 2% of SDS to spin to lyse the cells in 99°C for 5 minutes with 400μl each

Ø   Serial dilution of the bacteria of 2-fold, 4-fold, 8-fold and 16-fold was carried out

Ø   The sample (A-I) prepared by Frankie was added into the wells. 95μl each and a duplicate was done

Ø   Finally, 5μl of MQAE was added into the wells with the samples

Ø   The microplate was put into the incubator at 37°C for 1 hour.

3.          Measured the MQAE fluorescence by microplate reader.

4.          Following tubes are inoculated with DH5α pET27b

Ø   LB + IPTG

Ø   0.4M NaCl + IPTG without light

Ø   0.4M NaCl + IPTG with light

Ø   0.4M KCl + IPTG without light

Ø   0.4M KCL + IPTG with light

Ø   ( 10μl  K and 12.5μ of 0.4M of IPTG)

28 Aug (Sun)

1.      restriction cut of T7, HR (provided by Frankie) and plasmid 1C3

Ø   HR+T7:
- HR cut by Xbal1 & Pst1
- T7 cut by EcoR1 & Spe1

Ø   HR+1C3, T7+1C3: HR, T7, 1C3 cut by EcoR1 & Pst1

2.      ligation of HR & T7, HR & 1C3 backbone, T7 and 1C3 backbone respectively

Ø   10X T4 ligase buffer (2ul) , T4 ligase (1ul) for all tubes

Ø   HR+T7: HR (8.5ul), T7(8.5ul)
- 2 sets, 1 set at 16^oC overnight, another set at room temperature, 1hour

Ø   HR+1C3
- 2 sets, 1 set at 16^oC overnight, another set at room temperature, 1hour

Ø   T7+1C3
- 1set, at 16^oC overnight

Ø   Aliquot 3 tubes with 30ul 10X T4 ligation buffer

Ø   Use 10X T4 ligation buffer in 1.5ml microcentrifuge tubes

Week 13 Monday 29 Aug – Sunday 4 Sept

29Aug (Mon)

1. PCR purification of ligation product

2. Run gel

Lane	1	2	3	4	5	6	7
Sample	1bb ladder	100bp ladder	HR T7 @16^oC	HR T7 @22^oC	T7 1C3 @16^oC	HR 1C3 @22^oC	HR 1C3 @16^oC

3. Nanodrop

4. Transformation

Ø Use DH5a and BL21 as competent cells for each set

Ø T71C3 @16^oC

Ø HR1Ce@16^oC

Ø HR1C3@22^oC

Ø 6 spread plate stored at incubator

5. Following tubes are inoculated with DH5α pET27b

Ø LB + IPTG

Ø 0.4M NaCl + IPTG without light

Ø 0.4M NaCl + IPTG with light

Ø 0.4M KCl + IPTG without light

Ø 0.4M KCL + IPTG with light

Ø ( 10μl K and 12.5μ of 0.4M of IPTG)

30Aug (Tue)

1. PCR purification of HR1C3(16^oC), T71C3(16^oC), T7HR(16^oC)

2. Restriction cut wth

Ø T7-HR: Spe1

Ø T7-HR: EcoR1 & Pst1

Ø 24C double terminator: Xbal1 & Pst1

Ø pSB1C3: EcoR1 & Pst1

3. PCR amplify and purify: HR, T7, HR-T7

4. Run gel to check size

Ø Result:
-HR, T7 à correct bands
-HR-T7 à no band

5. Nanodrop of step 4 product

Sample	ng/ul	260/280
HR	12.4	1.70
T7	7.9	1.99
HR-T7	8.0	1.57

6. Ligation of step 2 restriction cut product

Ø (T7-HR) + (24C double terminator)

Ø (T7-HR) + (pSB1C3)

7. Spread plate

Ø HR1C3 DH5a 16^oC

Ø HR1C3 BL21 22^oC

Ø T71C3 DH5a 16^oC

Ø T71C3 BL21 16^oC

Ø Chloride sensor

8. Measured the OD600 of the bacteria

Set up	LB + IPTG without light	NaCl + IPTG with light	NaCl + IPTG without light	KCl + IPTG with light	KCl + IPTG without light
OD₆₀₀(2X)	0.946	0.264	0.302	0.359	0.631
Volume to bespinned down (ml)	2.5	9.0	7.9	6.6	3.8

9. Use 2% of SDS to spin to lyse the cells in 99°C for 5 minutes with 400μl each

Ø Serial dilution of the bacteria of 2-fold, 4-fold, 8-fold and 16-fold was carried out

Ø The sample (A-I) prepared by Frankie was added into the wells

Ø 95μl each and a duplicate was done

Ø Finally, 5μl of MQAE was added into the wells with the samples

Ø The microplate was put into the incubator at 37°C for 1 hour.

10. Measured the MQAE fluorescence by microplate reader.

11. Following tubes are inoculated with DH5α pET27b

Ø LB + IPTG

Ø 0.4M NaCl + IPTG without light

Ø 0.4M NaCl + IPTG with light

Ø 0.4M KCl + IPTG without light

Ø 0.4M KCL + IPTG with light

Ø ( 10μl K and 12.5μ of 0.4M of IPTG)

31Aug (Wed)

1. PCR purification of ligation product

Ø (T7-HR)

Ø (T7-HR) + (24C double terminator)

Ø (T7-HR) + (pSB1C3)

2. Run gel

Ø (T7-HR) , (T7-HR) + (24C double terminator)àno band

Ø (T7-HR) + (pSB1C3)àwrong size

3. Restriction cut and ligation

Ø (T7-HR), HR1C3, T71C3

4. Run gel with ligation sample

Ø HR1C3àcorrect size

Ø T7-HRàno band

5. Gel recovery with HR1C3

6. Pick colony from plate prepared last night

Ø T71C3: DH5a

Ø HR1C3: DH5a & BL21

7. PCR amplification of HR & T7

8. Measured the OD600 of the bacteria

Set up	LB + IPTG without light	NaCl + IPTG with light	NaCl + IPTG without light	KCl + IPTG with light	KCl + IPTG without light
OD₆₀₀(2X)	0.301	0.588	0.454	0.269	0.468
Volume to bespinned down (ml)	8	4.1	5.3	9	5.2

9. Use 2% of SDS to spin to lyse the cells in 99°C for 5 minutes with 400μl each

Ø Serial dilution of the bacteria of 2-fold, 4-fold, 8-fold and 16-fold was carried out

Ø The sample (A-I) prepared by Frankie was added into the wells

Ø 95μl each and a duplicate was done

Ø Finally, 5μl of MQAE was added into the wells with the samples

Ø The microplate was put into the incubator at 37°C for 1 hour.

10. Measured the MQAE fluorescence by microplate reader.

1 Sept (Thu)

1. Nanodrop

a. PCR amplified HR & T7

Sample	ng/ul	260/280
HR	5.4	1.83
T7	27.1	1.73

b. Ligation

Sample	ng/ul	260/280
HR1C3 @16^oC (1)	16.8	1.76
(2)	18.6	1.76
(3)	12.8	1.79
(4)	16.1	1.78
HR1C3 @22^oC (1)	5.7	1.96
(2)	6.9	1.69
(3)	5.2	1.77
(4)	7.5	1.64

c. Gel recovery

Ø HR1C3 gel clean: 2.0ng/ul

2. Run gel to check size

Ø PCR amplified HR & T7 àboth correct

Ø HR1C3, T71C3àwrong size

Ø HR-T7àno band

3. Restriction cut: HR, T7, pSB1K3

Ø HR, T7, 1C3: EcoR1 & Pst1

Ø HR: Xbal1

Ø T7: Spe1

4. Ligation

Ø HR1K3

Ø T71K3

Ø HR-T7

Ø 16^oC, overnight

5. Transformation of gel clean HR1C3 to DH5a

Ø Spread 3 plates

2 Sept (Fri)

1. Transfer lab

2. Pick colony from plate prepared on 1/9

3. Prepared and autoclaved the following solutions

Ø 500ml LB solution

Ø 250ml LB solution with 1M NaCl

Ø 250ml LB solution with 1M KCl

3 Sept (Sat)

1. Miniprep of DH5a with HR1C3

2. Run gel with miniprep product

Week 14 Monday 5 Sept – Sunday 11 Sept

5 Sept (Mon)

1. Transformation of chloride sensor and 24C terminator

2. Nanodrop of HR1C3 prepared on 3/9

sample	ng/ul	260/280
1	46.6	1.85
2	60.5	1.81
3	58.9	1.88

6 Sept (Tue)

1. Restriction cut: HR1K3, T71K3, T7HR1K3, pSB1C3

Ø HR1K3, T71K3, T7HR1K3, pSB1C3: EcoR1 & Pst1

2. PCR Purification

3. Ligation

Ø HR+pSB1C3

Ø T7+pSB1C3

Ø T7-HR+pSB1C3

4. Pick colonies of 24C terminator

7 Sept (Wed)

1. Transformation of HR1C3, T71C3 & T7HR1C3 into DH5alpha

2. Miniprep 24C terminator (low concentration)

8 Sept (Thu)

1. Pick Colonies of HR1C3 & T7HR1C3, T71C3 have no colonies

2. Restriction cut T71K3 and pSB1C3

3. Ligation T7+1C3

4. Transformation of 24C terminator

9 Sept (Fri)

1. Miniprep HR1C3 and T7HR1C3

2. Transformation of T71C3 into DH5alpha

3. Pick colonies of 24C terminator

10 Sept (Sat)

1. T71C3 no colonies

2. Miniprep 24C terminator

12 Sept (Mon)

1. Restriction cut T71K3 and pSB1C3

2. PCR Purification

3. Ligation T7+1C3

13 Sept (Tue)

1. Transformation of T71C3 into DH5alpha

2. Transformation of HR1C3 and T7HR1C3 into DH5alpha for amplification

14 Sept (Wed)

1. Restriction cut 24C1AK3 and T7HR1C3

Ø T7HR1C3: EcoR1 & Spe1

Ø 24C1AK3: Xba1 & Pst1

2. PCR Purification

3. Ligation T7HR + 24C + 1C3

4. Pick colonies of HR1C3 and T7HR1C3

15 Sept (Thu)

1. Miniprep HR1C3 and T7HR1C3

2. Transformation of T7HR24C1C3

3. Characterize the cell adhersion by micromagnetic particles and movement undergone magnetic field attraction as a proof-of-concept model in tubes

16 Sept (Fri)

1. T7HR24C1C3 no colonies

2. Restriction cut T7HR1C3, 24C1AK3 & pSB1T3

Ø T7HR1C3: EcoR1 and Spe1

Ø 24C1AK3: Xba1 and Pst1

Ø pSB1T3: EcoR1 and Pst1

3. PCR purification

4. Ligation T7HR+24C+1T3

17 Sept (Sat)

1. Transformation of T7HR24C1T3

18 Sept (Sun)

1. T7HR24C1T3 no colonies

2. PCR amplification of T7HR & 24C double terminator using a pair of primers respectively

3. PCR purification

4. Restriction cut T7HR, 24C and pSB1C3

Ø T7HR: EcoR1 and Spe1

Ø 24C: Xba1 and Pst1

Ø pSB1C3: Ecor1 and Pst1

5. Run gel and gel recovery

6. Ligation T7HR + 24C + pSB1C3

19 Sept (Mon)

1. Transformation of T7HR24C1C3

2. Restriction cut of T7HR1C3 and 24C1AK3

Ø T7HR1C3: Ecor1 and Spe1

Ø 24C1AK3: Xba1 and Pst1

3. PCR Purified T7HR1C3 cut

4. Run gel and gel recovery of 24C cut

5. Ligation T7HR24C1C3

20 Sept (Tue)

1. Pick colonies of T7HR24C1C3 from PCR amplification

2. Transformation of T7HR24C1C3 from standard assembly

3. Characterize the cell adhersion by micromagnetic particles and movement undergone magnetic field attraction as a proof-of-concept model in tubes (repeat of 15 Sept)

21 Sept (Wed)

1. Miniprep T7HR24C1C3 from PCR amplification (low concentration)

2. Pick colonies of T7HR24C1C3 from standard assembly

22 Sept (Thu)

1. Miniprep T7HR24C1C3 from standard assembly

2, Run gel check size of T7HR24C1C3

3. Do DNA sequencing on the biobricks we want to submit

Lane 1: 1kb ladder

Lane 2: HR1C3 cut by EcorR1 and Pst1

Lane3&4: T7HR24C1C3 cut by Ecor1 and Pst1

Lane6&7: T7HR1C3 cut by Ecor1 and Pst1

Results: correct size of T7HR24C1C3 biobricks Successfully inserted the 24C double terminator.

23 Sept (Fri)

1. Characterize if biobricks performance same as theoretical HR gene performance on a) Cl- pumping b) light absoption efficiency c) regulated protein expression level

2. Characterize the cell movement undergone magnetic field attraction in a small-scale proof-of-concept design (repeat of 15 Sept, except same scale as power generation system is being used to demonstrate its movement rate)

3. Receive the DNA sequence file of biobricks BBa_K559000, BBa_K559001, the sequencing results are correct

Series of data has been put in part registry and wiki on our biobricks BBa_K559000 and BBa_K559010.

24 Sept (Sat)

1. Characterize the cell movement undergone magnetic field attraction to be filmed undergone UV light and microscopy

Video and photos of the cell movement have been filmed

25 Sept (Sun)

1. Repeat characterization of the biobricks performance of 23 Sept

2. Characterize the cell movement undergone magnetic field

Result: All the data are consistent with the work on 23 Sept

26 Sept (Mon)

1. Repeat characterization of the biobricks performance of 25 Sept

2. Receive the DNA sequence file of biobricks BBa_K559010 , the sequencing result is correct

3. Do size check of biobricks BBa_K559010 again to confirm its size correctness

Result: All the data are consistent with the work on 25 Sept, we now have 3 sets of independent results to make error bars for characterization

Lane 1: 1kb ladder

Lane 3: T7HR24C1C3HR1C3 cut by EcoR1 and Pst1

Lane 5: HR1C3 cut by EcoR1 and Pst1

We now have correct biobricks to be sent to iGEM part registry!!!

27 Sept (Tue)

1. Pack all the biobrick samples into iGEM submission format

2. Prepare additional sample by doing bacterial transformation to get more DNA of our correct biobricks in case we need it

28 Sept (Wed)

1. Do series of inoculation, miniprep to get more of our biobricks BBa_K559001, BBa_K559001, BBa_K559010 and nanodrop to measure our biobricks to ensure all the biobricks have enoughamount to store in our lab for future use

??

"Creativity is thinking up new things. Innovation is doing new things." - Theodore Levitt

@@ Line 8,274: / Line 8,274: @@
 </table>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><b>&nbsp;</b></div>
-<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><b><span style="font-size: 13pt; "><img width="731" height="435" v:shapes="C_x0020_9" alt="" src="http://samlo.t0.ru/igem/(3b)Laboratory_Log%20book_JackyFinal_files/image006.gif" /></span></b></div>
+<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><b><span style="font-size: 13pt; "><img width="731" height="435" v:shapes="C_x0020_9" alt="" src="http://samlo.t0.ru/igem/stock/image006.gif" /></span></b></div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; ">&nbsp;</div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; ">&nbsp;</div>
@@ Line 8,452: / Line 8,452: @@
 </table>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; ">&nbsp;</div>
-<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><img width="668" height="413" v:shapes="C_x0020_10" alt="" src="http://samlo.t0.ru/igem/(3b)Laboratory_Log%20book_JackyFinal_files/image008.gif" /></div>
+<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><img width="668" height="413" v:shapes="C_x0020_10" alt="" src="http://samlo.t0.ru/igem/stock/image008.gif" /></div>
-<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><img width="677" height="415" v:shapes="C_x0020_11" alt="" src="http://samlo.t0.ru/igem/(3b)Laboratory_Log%20book_JackyFinal_files/image010.gif" /></div>
+<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><img width="677" height="415" v:shapes="C_x0020_11" alt="" src="http://samlo.t0.ru/igem/stock/image010.gif" /></div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; ">&nbsp;</div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; ">&nbsp;</div>
@@ Line 8,810: / Line 8,810: @@
 </table>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><u>Result</u></div>
-<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><img width="671" height="410" v:shapes="C_x0020_12" alt="" src="file:///P:/Desktop/xxif3/http://samlo.t0.ru/igem/(3b)Laboratory_Log%20book_JackyFinal_files/image012.png" /></div>
+<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><img width="671" height="410" v:shapes="C_x0020_12" alt="" src="http://samlo.t0.ru/igem/stock/image012.png" /></div>
 <div style="margin-left: 48pt; text-indent: -24pt; "><span style="font-family: Wingdings; ">&Oslash;<span style="font: normal normal normal 7pt/normal 'Times New Roman'; ">&nbsp;&nbsp;&nbsp;</span></span>Brian suggested that the abnormality of the cell count data might be due to the growing size of the bacteria. At 3.0 hour, signal could not be obtained as well as the reason.</div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; ">&nbsp;</div>
@@ Line 8,917: / Line 8,917: @@
 <div style="margin-left: 42pt; text-indent: -24pt; "><span style="font-family: Wingdings; ">&Oslash;<span style="font: normal normal normal 7pt/normal 'Times New Roman'; ">&nbsp;&nbsp;&nbsp;</span></span>1mM MQAE</div>
 <div style="margin-left: 0.25in; text-indent: -0.25in; "><b><span>5.<span style="font: normal normal normal 7pt/normal 'Times New Roman'; ">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></span></b><b>95</b><b><span style="font-family: Cambria, serif; ">&mu;</span>l of&nbsp;NaCl&nbsp;solutions and 5&nbsp;</b><b><span style="font-family: Cambria, serif; ">&mu;l</span></b><b>&nbsp;of MQAE solutions were added into the&nbsp;microplate&nbsp;in the position as shown below.</b></div>
-<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><span style="position: relative; z-index: 251660288; left: -49px; top: 25px; width: 700px; height: 386px; "><img width="700" height="361" v:shapes="_x0000_s1030 _x0000_s1026 _x0000_s1027 _x0000_s1028 _x0000_s1029" alt="" src="file:///P:/Desktop/xxif3/http://samlo.t0.ru/igem/(3b)Laboratory_Log%20book_JackyFinal_files/image013.png" /></span>&nbsp;</div>
+<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><span style="position: relative; z-index: 251660288; left: -49px; top: 25px; width: 700px; height: 386px; "><img width="700" height="361" v:shapes="_x0000_s1030 _x0000_s1026 _x0000_s1027 _x0000_s1028 _x0000_s1029" alt="" src="http://samlo.t0.ru/igem/stock/image013.png" /></span>&nbsp;</div>
 </div>
 <div><span style="font-size: 12pt; font-family: Calibri, sans-serif; "><br clear="all" style="page-break-before: always; " />
@@ Line 10,817: / Line 10,817: @@
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 21.3pt; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; text-indent: -0.25in; "><b><span>3.<span style="font: normal normal normal 7pt/normal 'Times New Roman'; ">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></span></b><b>PCR with T7 &amp; HR</b></div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 21.3pt; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; text-indent: -0.25in; "><b><span>4.<span style="font: normal normal normal 7pt/normal 'Times New Roman'; ">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></span></b><b>PCR purification of PCR product of step 3</b></div>
-<div style="margin-top: 0in; margin-right: 0in; margin-left: 3.3pt; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><b><img width="320" height="240" v:shapes="_x0000_i1031" alt="" src="file:///P:/Desktop/xxif3/http://samlo.t0.ru/igem/(3b)Laboratory_Log%20book_JackyFinal_files/image014.jpg" /></b></div>
+<div style="margin-top: 0in; margin-right: 0in; margin-left: 3.3pt; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><b><img width="320" height="240" v:shapes="_x0000_i1031" alt="" src="http://samlo.t0.ru/igem/stock/image014.jpg" /></b></div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 21.3pt; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; text-indent: -0.25in; "><b><span>5.<span style="font: normal normal normal 7pt/normal 'Times New Roman'; ">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></span></b><b>Measured the OD600 of the back up set of the bacteria</b></div>
 <table border="1" cellspacing="0" cellpadding="0" style="border-collapse: collapse; border-top-style: none; border-right-style: none; border-bottom-style: none; border-left-style: none; border-width: initial; border-color: initial; ">
@@ Line 10,893: / Line 10,893: @@
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0.25in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; text-indent: -0.25in; "><span>8.<span style="font: normal normal normal 7pt/normal 'Times New Roman'; ">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></span>T7: result ok, at 100-200bp</div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; ">&nbsp;</div>
-<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><img width="320" height="240" v:shapes="_x0000_i1032" alt="" src="file:///P:/Desktop/xxif3/http://samlo.t0.ru/igem/(3b)Laboratory_Log%20book_JackyFinal_files/image015.jpg" />&nbsp;</div>
+<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><img width="320" height="240" v:shapes="_x0000_i1032" alt="" src="http://samlo.t0.ru/igem/stock/image015.jpg" />&nbsp;</div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; ">&nbsp;</div>
-<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><img width="320" height="240" v:shapes="_x0000_i1033" alt="" src="file:///P:/Desktop/xxif3/http://samlo.t0.ru/igem/(3b)Laboratory_Log%20book_JackyFinal_files/image016.jpg" /></div>
+<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><img width="320" height="240" v:shapes="_x0000_i1033" alt="" src="http://samlo.t0.ru/igem/stock/image016.jpg" /></div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; ">&nbsp;</div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; ">&nbsp;</div>
@@ Line 11,354: / Line 11,354: @@
      </tbody>
 </table>
-<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><img width="575" height="286" v:shapes="C_x0020_1" alt="" src="file:///P:/Desktop/xxif3/http://samlo.t0.ru/igem/(3b)Laboratory_Log%20book_JackyFinal_files/image018.png" /></div>
+<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><img width="575" height="286" v:shapes="C_x0020_1" alt="" src="http://samlo.t0.ru/igem/stock/image018.png" /></div>
-<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><img width="623" height="334" v:shapes="C_x0020_2" alt="" src="file:///P:/Desktop/xxif3/http://samlo.t0.ru/igem/(3b)Laboratory_Log%20book_JackyFinal_files/image020.png" /></div>
+<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><img width="623" height="334" v:shapes="C_x0020_2" alt="" src="http://samlo.t0.ru/igem/stock/image020.png" /></div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 21.3pt; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; text-indent: -0.25in; "><b><span>7.<span style="font: normal normal normal 7pt/normal 'Times New Roman'; ">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></span></b><b>2% of SDS was spin to lyses the cells in 99&deg;C for 5 minutes with 400&mu;l each</b></div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 45.3pt; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; text-indent: -24pt; "><span style="font-family: Wingdings; ">&Oslash;<span style="font: normal normal normal 7pt/normal 'Times New Roman'; ">&nbsp;&nbsp;&nbsp;</span></span>Serial dilution of the bacteria in 8X, 16X, 32X, 64X and of salt like previously done was carried out</div>
@@ Line 11,444: / Line 11,444: @@
      </tbody>
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-<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><span style="font-family: 新細明體, serif; "><img width="599" height="322" v:shapes="C_x0020_3" alt="" src="file:///P:/Desktop/xxif3/http://samlo.t0.ru/igem/(3b)Laboratory_Log%20book_JackyFinal_files/image022.png" /></span></div>
+<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><span style="font-family: 新細明體, serif; "><img width="599" height="322" v:shapes="C_x0020_3" alt="" src="http://samlo.t0.ru/igem/stock/image022.png" /></span></div>
-<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><span style="font-family: 新細明體, serif; "><img width="599" height="358" v:shapes="C_x0020_4" alt="" src="file:///P:/Desktop/xxif3/http://samlo.t0.ru/igem/(3b)Laboratory_Log%20book_JackyFinal_files/image024.png" /></span></div>
+<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><span style="font-family: 新細明體, serif; "><img width="599" height="358" v:shapes="C_x0020_4" alt="" src="http://samlo.t0.ru/igem/stock/image024.png" /></span></div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 21.3pt; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; text-indent: -0.25in; "><b><span>5.<span style="font: normal normal normal 7pt/normal 'Times New Roman'; ">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></span></b><b>2% of SDS was&nbsp;<span>spinned&nbsp;to lyse the cells in 99&deg;C for 5 minutes with 400&mu;l each</span></b></div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 45.3pt; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; text-indent: -24pt; "><span style="font-family: Wingdings; ">&Oslash;<span style="font: normal normal normal 7pt/normal 'Times New Roman'; ">&nbsp;&nbsp;&nbsp;</span></span>Serial dilution of the bacteria in 8X, 16X, 32X, 64X and of salt like previously done was carried out</div>
@@ Line 11,758: / Line 11,758: @@
      </tbody>
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-<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><img width="629" height="339" v:shapes="_x0000_i1038" alt="" src="file:///P:/Desktop/xxif3/http://samlo.t0.ru/igem/(3b)Laboratory_Log%20book_JackyFinal_files/image026.png" /><img width="623" height="358" v:shapes="_x0000_i1039" alt="" src="file:///P:/Desktop/xxif3/http://samlo.t0.ru/igem/(3b)Laboratory_Log%20book_JackyFinal_files/image028.png" /></div>
+<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; "><img width="629" height="339" v:shapes="_x0000_i1038" alt="" src="http://samlo.t0.ru/igem/stock/image026.png" /><img width="623" height="358" v:shapes="_x0000_i1039" alt="" src="http://samlo.t0.ru/igem/stock/image028.png" /></div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; text-indent: 0in; "><b><span>2.<span style="font: normal normal normal 7pt/normal 'Times New Roman'; ">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></span></b><b>Use 2% of SDS to spin to lyse the cells in 99&deg;C for 5 minutes with 400&mu;l each</b></div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 45.3pt; margin-bottom: 0.0001pt; font-size: 12pt; font-family: Calibri, sans-serif; text-indent: -24pt; "><span style="font-family: Wingdings; ">&Oslash;<span style="font: normal normal normal 7pt/normal 'Times New Roman'; ">&nbsp;&nbsp;&nbsp;</span></span>Serial dilution of the bacteria of 2-fold, 4-fold, 8-fold and 16-fold was carried out</div>
@@ Line 11,974: / Line 11,974: @@
 -HR-T7&nbsp;<span>&agrave;</span>&nbsp;no band</div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; ">&nbsp;</div>
-<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><img width="320" height="240" v:shapes="_x0000_i1040" alt="" src="file:///P:/Desktop/xxif3/(3b)Laboratory_Log%20book_JackyFinal_files/image029.jpg" /></div>
+<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><img width="320" height="240" v:shapes="_x0000_i1040" alt="" src="(3b)Laboratory_Log%20book_JackyFinal_files/image029.jpg" /></div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 21.3pt; margin-bottom: 0.0001pt; font-size: 12pt; text-indent: -0.25in; "><b><span>5.<span style="font: normal normal normal 7pt/normal 'Times New Roman'; ">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</span></span></b><b><span>Nanodrop</span></b><b>&nbsp;of step 4 product</b></div>
 <table border="1" cellspacing="0" cellpadding="0" style="margin-left: 21.3pt; border-collapse: collapse; border-top-style: none; border-right-style: none; border-bottom-style: none; border-left-style: none; border-width: initial; border-color: initial; ">
@@ Line 12,526: / Line 12,526: @@
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b>Lane6&amp;7: T7HR1C3 cut by Ecor1 and Pst1</b></div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b>Results: correct size of T7HR24C1C3&nbsp;<span>biobricks&nbsp;Successfully&nbsp;inserted the 24C double terminator.</span></b></div>
-<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b><img width="516" height="784" v:shapes="_x0000_i1041" alt="" src="file:///P:/Desktop/xxif3/(3b)Laboratory_Log%20book_JackyFinal_files/image030.jpg" /></b></div>
+<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b><img width="516" height="784" v:shapes="_x0000_i1041" alt="" src="(3b)Laboratory_Log%20book_JackyFinal_files/image030.jpg" /></b></div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b>&nbsp;</b></div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b><u>23 Sept (Fri)</u></b></div>
@@ Line 12,559: / Line 12,559: @@
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b>Lane 5: HR1C3 cut by EcoR1 and Pst1</b></div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b>&nbsp;</b></div>
-<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b><img width="311" height="307" v:shapes="_x0000_i1042" alt="" src="file:///P:/Desktop/xxif3/(3b)Laboratory_Log%20book_JackyFinal_files/image032.jpg" /></b></div>
+<div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b><img width="311" height="307" v:shapes="_x0000_i1042" alt="" src="(3b)Laboratory_Log%20book_JackyFinal_files/image032.jpg" /></b></div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b>&nbsp;</b></div>
 <div style="margin-top: 0in; margin-right: 0in; margin-left: 0in; margin-bottom: 0.0001pt; font-size: 12pt; "><b>&nbsp;</b></div>

Solution (added by order)	Amount(ul)
ddH₂0	14
10X Buffer 3	2.5
10X BSA	2.5
EcoR1	0.5
Pst1	0.5
HR	5
Total	25ul

[Salt]/M	0	0.1	0.2	0.3	0.4	0.5	0.6	0.7	0.8	0.9	1.0
LB + salt/ml	0	0.4	0.8	1.2	1.6	2.0	2.4	2.8	3.2	3.6	4
LB/ml	4.0	3.6	3.2	2.8	2.4	2.0	1.6	1.2	0.8	0.4	0

H2O	11ul
10X T4 DNA ligase buffer	2ul
T4 DNA ligase	1ul
HR	2ul
T7	2ul
1A3	2ul
Total	20ul

Start culture	LB	KCl 0.4M	NaCl 0.4M	KCl 0.6M	NaCl 0.6M
First measurement (4X)	0.430	0.674	0.410	0.380
Second measurement (4X)	0.401	0.636	0.431	0.488	0.348

Time (h)/ culture	LB	KCl 0.4M	NaCl 0.4M	KCl 0.6M	NaCl 0.6M
0	0.023	0.046	0.027	0.022	0.025
0.5	0.025	0.038	0.029	0.029	0.027
1.0	0.036	0.032	0.026	0.039	0.048
1.5	0.078	0.038	0.035	0.043	0.041
2.0	0.142	0.104	0.057	0.038	0.040
2.5	0.295	0.154	0.113	0.053	0.053
3.0	0.437	0.287	0.179	0.077	0.067
3.5	0.601	0.408	0.270	0.126	0.104
4.0	0.796	0.580	0.373	0.162	0.146

Time (h)	OD₆₀₀	Cell Count (particle per ml)
0	-0.004	4.5 X 10⁸
0.5	-0.002	5.2 X 10⁸
1.0	±0.000	6.7 X 10⁸
1.5	+0.002	8.7 X 10⁹
2.0	+0.007	1.8 X 10⁹
2.5	+0.026	1.8 X 10⁹
3.0	+0.041	3.1 X 10⁹
3.5	+0.061	4.8 X 10⁹

HR	7ul
T7	7ul
1G	2ul
Ligation buffer	2ul
T4 ligase	1ul
H2O	1ul
TOTAL	20ul

Mass (mg)	100
Molar mass (g mol^-1 )	326.1893
Concentration (mM)	100
Volume made (ml)	3.066

	ng/ul	260/280
3-way ligation	11.5	1.83
HR-T7-1T3 (1)	13.9	1.86
HR-T7-1T3 (2)	13.2	1.91
T71T3 (1), prepared on 6/8	29.4	1.52
T71T3 (2) , prepared on 6/8	7.3	1.69
T71T3 (3) , prepared on 6/8	5.4	1.74

Buffer 2	2ul
BSA	0.2ul
XBa 1	0.5ul
Pst 1	0.5ul
HR1K3 (prepared on 4/8)	16.8ul
Total	20ul
Buffer 2	2ul
BSA	0.2ul
EcoR1	0.5ul
Pst 1	0.5ul
3A (prepared on 4/8)	2ul
H2O	14.8ul
Total	20ul

T4 ligase buffer	2ul
T4 ligase	1ul
T7	7ul
HR	7ul
Vector pSB1C3 from 3-way	3ul
Total	20ul

	LB of 0.4M NaClwith light	LB of 0.4M NaClwithout light	LB with light	LB without light
OD₆₀₀(2X)	0.328	0.503	0.875	0.934

Tube	Concentration (ug/ul)	260/280
3A Post mini prep (1)	96.1	1.91
3A Post mini prep (2)	41.6	1.96
3A Post mini prep (3)	100.5	1.85
3A (1)	104.7	1.85
3A (2)	68.8	1.87
3A (3)	74.7	1.96
T71T3 (1)	87.3	1.79
T71T3 (2)	115.8	1.85
T71T3 (3)	135.9	1.91

	ug/ul	260/280
24C (1)	10.5	1.72
24C (2)	7.9	1.78
24C (3)	8.2	1.73
24C (4)	7.9	1.59
24C (5)	11.7	1.54
24C (6)	26.7	1.37
HR (1)	6	1.67
HR (2)	4.9	1.80
HR (3)	8	1.07

sample	Conc. (ng/ul)	260/280
HR1C3 (1)	30.5	1.74
(2)	24.7	1.81
(3)	19.9	1.94
(4)	22.1	1.97
(5)	28.3	1.80
(6)	50.0, 34.8, 21.0, 24.8, 25.3	1.94, 1.77, 2.25, 1.97, 1.91
(7)	42.1, 29.5	1.91, 1.92
(8)	24.6	1.78
(9)	31.4, 25.8	1.80, 1.91
(10)	18.9	1.91
(11)	19.2	1.99
(12)	48.2, 43.1, 45.8	1.88, 1.99, 1.92