Team:Hong Kong-CUHK/Notebook

From 2011.igem.org

2011 CUHK iGEM Note Book

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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 37oC , 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 MaCl24H2O (5.4425g)
2.      15mM CaCl22H2O (1.1025g)
3.      250mM KCl (9.3189g)
4.      10mM PIPES (10ml) (taken from 4oC fridge)
5.      Stored in 4oC 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 -80oc
 
 
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 37oC 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, 37oC 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 37oC, 2hrs
Ø   Remaining samples are stored at -20oC
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 at37oC 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 25oC 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 37oC, 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 -20oC
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 37oC for 2hrs
5.      Gel extraction of gel with 1T3 backbone from red conlony
Ø   4 tubes of backbone are made
Ø   Store at -20oC
6.      Pick red colony of DH5a
Ø   4tubes of LB snap cap
Ø   Incubate at 37oC, 25rpm
7.      Transfer to 125ml LB
Ø   Shake at 37oC 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 37oC, 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
-          5A pSB1K3 23/7
-          7A pSB1T3 23/7
-          T7-1K3 23/7
-          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 4oC
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 37oC
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 -16oC 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
-37oC, 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, 37oC, 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, 37oC 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   37oC, 2hrs
4.      Ligation
T4 ligase buffer
2ul
T4 ligase
1ul
T7
7ul
HR
7ul
Vector pSB1C3 from 3-way
3ul
Total
20ul
Ø   16oC overnight
5.      Measured OD600 of 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:
OD600 of 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