Team:Rutgers/LB1

From 2011.igem.org

Rutgers 2011 iGEM Team: Complex Circuits in Synthetic Biology

Rutgers 2011 iGEM Team: Complex Circuits in Synthetic Biology

RUTGERS iGEM TEAM WIKI

Laboratory Notebook

 

5/23/11 – Mini-Prep Procedure

Explained by – Kevin
Attended by – Steph, Sana, Ethan, Akanksha, Boris

Centrifuge (Angus Flexifuge) Overflows ano; Makes a mess with spin columns volume>500uL

Protocol 25 -  Preparation and Transformation of Competent E. Coli using CaCl2 (pg 1.116)
(Source – Molecular Cloning V1 – Seabrook and Russell)

Materials – CaCl2-2H20 (1M) or standard transformation buffer, MgCl2-CaCl2 solution (ice cold)

Media -  

  1. LB or SOB medium for initial growth of culture
  2. SOB agar plates containing 20uM MgSO4 and appropriate antibiotic
  3. SOC medium (appox. 1mL for each transformation)

Nuc. Acids – Plasmid DNA

Equipment –

  1. Polypropylene tube (50mL, chilled in ice)
  2. Polyprep tubes (17x100mm, chilled in ice)
  3. Water bath preset to 42 degrees

5/24/11
Tet+Amp Control had growth, Kan Control had no growth.
BB5 No growth.
Everything Else had growth.

Tet media was 10x too little Tet.
Recultured Tet controlled and transform into proper Tet.

Miniprepped and streaked Amp control and BB1.

Restriction of miniprep with:

 

Stock

1 Rxn

3 Rxn

XbaI

SpeI

ddH20

 

4.5uL

13.5uL

 

 

1x Buffer 4

10x

2uL

6uL

 

 

1x BSA .1mg/uL

10x

2uL

6uL

 

 

1 ug DNA

.1ug/uL

10uL

 

 

 

10 units XbaI

20000u/mL

.5uL

1.5uL

 

 

10 units SpeI

10000/u/mL

1uL

3uL

 

 

Total

 

20uL

60uL

 

 

1

2

3

4

5

6

7

8

9

10

L

L

 

MP2

R1

R2

 

 

 

 

5uL

10uL

 

 

 

 

 

 

 

 

MP1 – Miniprep BB1
MP2 – Miniprep Amp Control
R1 – Restricted MP1
R2 – Restricted MP2

Plated everything that came out
Future – If it looks good grow up and rerun restrictions with better spaced restriction sites.

5/25/11
All plates made on 5/24/11 grew into lawns

XbaI Digest
5uL H20
2uL buffer 4
2uL BSA
10uL DNA
1uL Enzyme (20 units) x2 samples
Then warm room 30 minutes.

The backbone for ES1 is pSBIAK8 which is 3426bp. The Brick for ES1 is 46bp, so the final plasmid should be around 3500bp.
XbaI cuts at 1272 and 2288 of backbone, which should give fragments of 1000bp and 2500bp (according to NEB cutter).
The gel is consistent with this if we assume MP1MP2

5/26/11
Results of seconds transformations

10am - AMP plates grew, no growth on Kan/Tet controls, no growth in Kan plate with ES5, and no growth in Tet plate ES2. Amp control has colonies (positive control with DNA)

1pm – Neil subcultured the two ccdB tubes that Kevin asked me to. They are labeled and in the roller. There is a mark on the tubes to indicate what tube was subcultured from what tube. We are taking 5 AMP plates and are going to perform streak-outs on them of the following plates – ES(1,3,4,6,7) from 5/25/11.

Streak-out plates were divided into 4 quadrants and we took four colonies from each plate.
Purpose – Separating individual colonies before additional miniprepping.

5/30/11
Experiment –
Overnights of ES(1,3,4,6,7) on LB+AMP, put in incubator (37 degrees C) at 1208pm. Single colony used is circled in place.

Colony counts from 5/25 transformation – TMTC=Too many to count


ES1

0

ES2

TMTC

ES3

TMTC

ES4

40

ES5

0

ES6

5

ES7

2

6/1/11
Protocol – Pouring Agar Plates

(from online source)
Moving Plates –
AMP – (1000xAMP); conc final – 100ug/mL. (100mg AMP/1000mL=100ug/mL)
Tet – (sus 1mL) – 2.5/5000mL = 50 sus/mL

LB Media
10g Bacto-tryptone (tryptone peptone)pancreatic digest of casein
5g Bacto-yeast extract (DFCO brand)
10g NaCl (red bp)
.5mL 10N NaOH (not necessary for plates)
For plates add 20g agar/litre
Autoclave 40’

AMP Plates
2 Liters LB Media made
20g tryp
10g yeast extract
20g NaCl
40g Agar/Litre

Tet Plates
5g Bacto-tryptone (tryptone peptone)
2.5 bacto yeast extract
5g NaCL (.5L)
For plates at 10g agar/litre

6/2/11
Made glycerol stocks of ccdB resistant-KanControl (600uL culture+600uL 80% glycerol)

Plated lovTAP stabs, streaked with toothpicks onto ChI plates.

Made ccdB resistant competent cells. Concentration of CaCl2 is not on bottle. Diluted overnight culture at 1:20 into 15mL LB at 1130am.

Made 100mm CaCl2 (stock 1M)
Calculations for 80mm MgCl2 20mM CaCl2 solution (MgCl2 stock 2M, CaCl2 stock 1M)

Final volume – 10mL
C1V1=C2V2, 1MV1=.02M10mL, 2MVa=.08M10mL, V1=.2mL CaCl2, Va=.4mL MgCl2, 9.4mL H20

Backbone Labels –
1I-4A5-SW
5C-2K3-MV
7c-3T5-AV

Transformations (4pm)
ES2 (Tet)
4A5 (Amp)
2K3 (Kan)
3T5 (Tet)
Amp+Control
Kan+Control
Amp-Control
Kan-Control
Tet-Control

Protocol –
Thaw comps on ice, aliquot 50uL per transformation, add 2uL DNA, incubate on ice for 20min
Add 200uL LB, incubate at 37 degrees C for 3 hours
Plate on antibiotic plates (50uL for amp, 100uL for kan/tet)

Notes – 200uL LB added at 430pm, cells plated at 700pm

6/6/11
ES3,4,5 PCR – all primers diluted to 100uM and then a tube of 10uM node.

For 50uL reaction volume x4
5uL 10x ThermoPol Buffer20uL
1ul 10(40mM) dNTPS4uL
1uL 10uM reverse primer4uL
1uL DNA
.5uL Vent2uL
1uL 100mM MgSO44uL
39.5 ddH2O158uL

DNA in kit is ~10mg resuspended in 10uL
50 1uL.001ug=1ng

Digestions – ES1,3,4,6,7 with Ecoli+PstI, also 8,9,C
Will use:
NEB Buffer 4 5uL/rxn45uL
100ug/mL BSA 5uL/rxn45uL
1uL EcoRI-HF 1uL/rxn9uL
1uL PstI 1uL/rxn9uL
50uL Reaction volume
5uL DNA 5uL/rxn
Total 17uL/rxn
H20 33uL/rxn297uL

Quick Purification of 5100L*1/250uLPH=2.0uL pH indicator

250uL buffer+50uL PCR
Buffer PB has accidentally too much pH indicator (6x too much) – may gel new kit

Digestions
Used 2uL enzyme - ES6EcoRI-HF+XbaI, ES7SpeI+PstI
Went in 20 minutes after first 2, used 3 uL Enzyme - PCR Purified ES3XbaI+PstI+DynI, PCR Purified ES4EcoRI-HF+SpeI+DpnI

All digestions done in NEB4 with BSA as yesterday

Ligations (x3)
Master mix
3.5uL H2O10.5uL
.5uL T4 Ligase1.5uL
1uL T4 Buffer3uL
1uL Vector
4uL Insert

Ligation Transformations 2uL on 48uL of comp cells, started incubation for 20mins at 346pm
PCRed at 405pm in incubator

6/8/11
Distribution kit contains extras of ES2. Took 1 and labeled it ES2-2. Found a possible alternative to ES5. Took it and labeled it ES5-2 (Brick#I12035). Also took VioABCE and VioABDE (Light and dark green respectively and labeled them ESLG and ESDG respectively.

Ran another transformation with grow up phase and some parts in IPTG.

ES5 – Kan+IPTG (added 20uL 100mM IPTG to each IPTG plate [20uL IPTG+80uL H2O)
ES5-2 – Kan+IPTG
ESDG – Kan
ESLG – Kan
ES2-2 – Tet
-Kan Control
+Kan Control
-Tet Control

Going to give 3hr grow up time at 37 degrees in LB (+IPTG for ES5, ES5-2, put in at 1145)

6/9/11
Miniprepped L3, L7D – KL
Digested with EcoRI-HF+PstI using previous protocol

1% agarose gel –
.8g agarose to 40mL of 1x TAE stock in 250mL bufferAdd 40mL 1x TAE Buffer to melted solutionAdd 5uL of 10mg/mL Ethidium Bromide

6/10/11
Remake Gel and rerun.

Digest (makes for 17 rxns)
NEB4 5uL/rxn85uL
100ug/mL BSA 5uL/rxn 85uL
EcoRI-HF 1uL/rxn17uL
PstI1uL/rxn17uL
H2O 33uL/rxn561uL
DNA 5uL/rxn

200pm 20 Digests+MP5Sill for 20 minutes at 80 degrees C 4 Dye

 

7/5
Digest L4 PCR with SyeI
50uL rxn vol
(x6.6)
5uLà33uL
5uLà33uL
1uLà6.6uL
10uLà66uL
29uLà191.4uL

37­oC @1217
50oC @1400

7/5
Gel
Digests coated 10uL
Rest coated 4uL

7/5
Started cultures of L9 and L6 in LB Amp

Redoing PCR of L4 minipreps
50uL rxn volume
(x2.2)
5uL 10x Thermopolà11uL
1uL 40um dNTPà2.2uL
1uL 10um VPZà2.2uL
1uL 10um VRà2.2uL
1uL DNA
.5uL ventà1.1uL
1uL 100um MgSO4à2.2uL
39.5uL ddH2Oà86.9uL

Dilute L4-C and LF-D 1:10
Use straight for A/B
Not using E/F

L6 PCR
50uL rxn vol
(x3.3)
1uL 40mm dNTPsà3.3uL
1uL 10um VF2à3.3uL
1uL 10um VRà3.3uL
1uL DNA
.5uL ventà1.65uL
1uL 100mm MgSO4à3.3uL
39.5uL ddH2Oà130.35
5uL 10x Thermopolà16.5


7/6
L6 PCR Gel
Gel shows PCR product between 200uL and 300bps which is bad.
Started liquid cultures of 6 more L6 colonies from transformation plate. Ignoring satellite colonies

7/7
Miniprepped new L6 Cultures

PCR of L4 (1-6) minipreps
50uL rxn volume
(x6.6)
5uL 10x thermopolà33uL
1uL 40mm dNTPsà6.6uL
1ul 10mm VP2à
1ul 10um VRà
1ul DNAà
.5ul ventà
1uL 100mM MgSO4à
39.5 ddH2Oà260.7uL

Sequencing (macrogen)
Primer made 2 pmol/k (?)(1:5 of PCR conc.)
Add 4uL primer to 10uL miniprep in strip tube
Label tubes 1-8
Tell Janet what 1-8 are (include primer)

PCR of L3-3 ES1, LtrpR-B, LtrpR-E
50uL
(x4.4)
5uL 10x thermpolà22
1uL 40mM dNTPsà4.4
1uL 10uM VP2à4.4
1uL 10uM VRà4.4
1uL DNA
.5uL vent à2.2
1uL 100mM MgSO4à4.4 uL
39.5 ddH2Oà150uL

L5 Digestions

Digest L4 PCR Product with ECORI, NheI, DpnI
Digest L3 PCR Product with XbaI, PstI, DpnI
Digest PSBIC3 Digestion with DpnI
50uL rxn vol
(2.2x)
5uL 10x NEB4à11uL
5uL 10x BSAà11uL
3uL enzyme
10uL DNA
27uL ddH2Oà59.4

For pSBIC3 system, just add 1uL DpnI to mix

L5 Ligation mix
21uL rxn Volume
2uL T4 LIgase Buffer
3uL L4 PCR Digest END
3uL L3 PCR D XPD
2uL pSBIC3 PCR D EPD
10uL ddH2O
1uL T4 Ligase

Transformations:
L5 (pSBIC3, L3, L4, PCR Digests)
EL7-2
(L3-3, L4-A, EAmp-Control, Ch1-Control)
Using standard protocol

7/12

50uL rxn vol
(2.2x)
5uL 10x thermopolà11.0
1uL 40mM dNTPsà2.2
1uL 10um VR2à2.2
1uL 10um VRà2.2
1uL DNA
.5 uL ventà1.1
1uL 100mM MgSO4à2.2
39.5 uL ddH2Oà86.9

Digest of L7, L3 (EX)
50uL rxn vol
5uL 10x NEB4
5uL 10x BSA
1uL EcoRI-HF
1uL XbaI
5uL DNA (A little short on L7)
33uL ddH2O

PCR Purifying L7/L3 digests since “inserts” are very small

Digest of L6, ES1, PCR with
50uL rxn vol
5uL 10x MB4
5uL BSA
1uL EcoRI-HD
1uL SpeI
1uL DpnI
5uL DNA
32uL ddH2O

7/12
Ligations 5,8

Ligation 8 – L6 Insert, L7 Backbone
Ligation 5 – L4 Insert, L3 Backbone

Ligation Mix –
20uL rxn vol
2uL 10x T4 ligase buffer
1uL T4 Ligase
1uL backbone
3uL insert
13uL ddH2O

DNA Heat shock – 5min

Transformations 5,8
50uL 5 and 8

Transformed L5 and L8 using standard protocol

ES7 minprep using standard protocol.

7/13
B1006-T1 plate 1 well  4H
B1006-T2 plate 1 well 4B
(both are pSB1AK3)

Transformed using standard protocol

ES4 positive control

Make glycerol stocks of L3-3, L4-A, L7-2, LtrpR-G, L6-2

Digestions – ES7-EX, ES7-SP, trpR-E PCR, XPD

50uL rxn vol
5uL NEB4
5uL BSA
1uL enzyme1
1uL enzyme 2
(1uL enzyme3)
10uL DNA
27uL ddH2O
(1uL ddH2O)
-37 degrees C
ES7 Digestions PCR Purified
trpR-E 80 degrees C

Ligations 9 and 11
20uL rxn vol
2uL T4 ligase buffer
1uL backbone
3uL insert
1uL T4 Ligase
13uL ddH2O

L9 insert ES1 DESD
Backbone ES7 DEX

L11 Insert trp-E PCR DXPD
Backbone ES7 DSP

Started liquid cultures of L5 and L8, 2 each

7/14
L5A-B, L8A-B miniprep using standard protocol

Miniprepped T1+T2 using standard protocol

7/14
Phusion PCR
98 degrees C 30 sec
(30 cycles of next 3)
98 degrees C 5 sec
63 degrees C 10 sec
72 degrees C 15 sec
72 degrees C 5 min
4 degrees C hold

50uL rxn vol
25uL phusion master mix
2.5uL VP2 primer
2.5uL VR Primer
1uL DNA
19uL dd H2O

Digest T2 with EX
50uL rxn vol
5uL 10x NEB4
5uL 10x BSA
1uL EcoRI-HF
1uL XbaI
5uL DNA
33uL ddH2O

 

7/14
T1 PCR digestion ESD
50uL rxn vol
5uL NEB4
5uL 10x BSA
1uL EcoRI-HF
1uL SpeI
1uL DpnI
10uL DNA
27uL ddH2O

L9 and L11 transformation – Colony count
Negative control – 0
Positive control – a lot
ES7 DSP – 41
trpR DX – 0
L9 ~5
ES1 PCR DES – 22
ES7 DEX – 37
L11 – 120

7/15
Miniprepped L11A-P and L9 1-4 using standard protocol

L9-2 messed up

7/18
PCR mRFP
50uL rxn vol
25uL 2x phusion master mix
2.5uL VR2 primer
2.5uL VR Primer
1uL DNA
19uL ddH2O

RBS digestion n with SP
(ES1)T7 with EX
50uL rxn bol
5uL NEB4
5uL 10x BSA
5uL DNA
23uL ddH2O
1uL enzyme 1
1uL enzyme 2

PCR of ES9
50uL rxn vol
25uL phusion 2x master mix
2.5uL VF2 primer
2.5uL primer
1uL DNA
19uL ddH2O

PCR purification RFP and ES9

ES9 and mRFP digestion
ES9 with ES
RFP with XP
50uL rxn vol
5uL NEB 4
5uL 10x BSA
1uL DPnI
10uL DNA
1uL enzyme 1
1uL enzyme 2
27uL H2O

trpR digestion with XP
50uL rxn vol
5uL NEB4
5uL 10x BSA
1uL PPNI
20uL DNA
1uL XbaI
1uL PstI
17uL H2O

7/18/11
Made LB/Chlorinphenacol plates and LB/Amp plates (2L each) using this procedure –
20g tryptone peptone
10g bacto-yeast 
20g NaCl
2L dH2O
40g Agar
Autoclave for 1 hour at 40

Phosphatone of ES7 and ES1
-take out 6mL of DNA
-add 5mL of phosphatase buffer
Add 1mL phosphatase
1hr @ 37 degrees C
5min@ degrees C

7/19
Ligations 18,19
20rxn vol
2uL T4 ligase buffer
1uL backbone insert
1uL T4 ligase
13uL ddH20

L18
8uL ES7 backbone
1uL mRFP insert

L19
3uL ES9 insert
1uL ES1 backbone

PCR L11 A-H
50rxn vol
25mL phusion master mix
2.5uL VF primer
2.5uL VR Primer
1uL DNA
19uL DNA

Transformed cells using standard protocol
Miniprepped ES7 using standard protocol

Colony PCR
20uL rxn vol
10uL ddH2O
10uL Phusion
1 Colony

Made cultures of L18 and L19 transformation

Transformation/# of colonies
L18 – 1
L19 – 2
+control – 8
ES7 DEX  -12
ES7 DSP – 2
-control – 0
mRFP – 0
ES9 DESD – 0

7/25
Buffer -
1g PEG 6000
.15mL 2M MgCl2
.5mL DMSO
9mL LB
pH to ~6.5
filter sterilize

L5 and T1 PCR digestion
L5 with SP
T1 with DXP
30 rxn vol
5uL NEB4
5uL 10XBSA
1uL DDNI
10uL DNA
1uL enzyme1
1uL enzyme2
27uL H2O

PCR LovTap
50uL rxn vol
25uL phusion buffer
22uL ddH2O
1uL DNA
1uL primer 1
1uL primer 2

Digested with JpnI
L11 done
L12 resequence
L17 bad because ptrpL in ES9 resequence

ES7 Digestion with SP
50uL rxn vol
5uL NEB4
5uL 10x BSA
1uL DNA
1uL enzyme1
1uL enzyme 2
28uL H2O
Kill @80 degrees C 20 minutes

Phosphatase of L5
50uL rxn vol
5uL phosphatase buffer
1uL phosphatase
44uL ddH2O
-1hr 37 degrees C
-5min kill 65 degrees C

Ligate L17
20uL rxn vol
2uL T4 Ligase buffer
1uL backbone
3uL Insert
1uL T4 Ligase
13uL ddH2O

Transformed L17 and L11
PCR L8 A1
50uL rxn vol
25uL 2x phusion master mix
2.5uL VF2 Primer
2.5uL VR Primer
1uL DNA
19uL H2O

L8A2 and ES7 digestion (L8A-2 and ES7-EX)
50uL rxn vol
5uL NEB4
5uL 10x BSA
10u DNA
1uL enzyme 1
1uL enzyme 2
28uL H2O

PCR Purify ES7 and L8A1
Phosphatase ES7 – 1hr @37 degrees C

L8A1 digestion DES
50uL rxn vol
5uL NEB4
5uL 10x BSA
10uL DNA
1uL enzyme 1
1uL enzyme 2
27uL ddH2O
1uL DPN1
80 degrees C Kill

ES7 Phos
L8A1 DDES

 

Ligations 3,7 (again)
PCR ES3, ES4 with VR, VF2
Rxn mix (5x)
5uL 10x bufferà25uL
1uL 40mm dNTP (10mm each)à5uL
.5uL 100um VF2à2.5uL
.5uL 100um VRà2.5uL
1uL plasmid (once with straight, once 1:10 dilution)
1uL Ventà5uL
40uLddH2Oà200uL
1 uL MgSO4à5uL

Thermocycler settings
95 degrees C 2 min
(30 cycles of the rest)
95 degrees C 30 sec
50.3 degrees C 30 sec
74.0 degrees C 1 min
74.0 degrees C 5min
4 degrees C hold

Digest of ES6, ES7
ES6 digested with EcoRI+XbaI
ES7 digested with  PstI+SpeI
50uL rxn vol
5uL 10x NEB buffer 4
5uL 10x BSA
1uL enzyme 1
1uL enzyme 2
10uL DNA (from miniprep)
28uL ddH2O

2 hour incubator at 37 degrees C
20 min incubator at 80 degrees C
Add 5uL Antarctic 10x buffer, 1uL Antarctic Phosphatase (Take 6uL aliquot for control-unphosphorylated)
1 hour incubation at 37 degrees C
20 min activationat 80 degrees C
Note – grey dry bath high ~4 give 80 degrees C

Ligations 3,7
PCR Purification of 40uL product as per qiagen kit. (extra 10uL will be used to check kit yield)

Digest of ES3, ES4 (PCR products)
ES3 digested with XbaI and PstI
ES4 digested with EcoRI-HF and SpeI

Digest mix
50uL rxn volume
5uL NEB4 (10x)
5uL 10x BSA
1uL enzyme 1
1uL enzyme 2
1uL DpnI
10uL DNA
27uL ddH2O

Incubate at 37 degrees C for 2 hours, make another set to digest for 1hr. inactivate at 80 degrees C for 20min

1% agarose gel 40 wells made.
80mL/TAE. 8g Agarose (gel is a bit thin, next time go for 100mL)

Ligations 3 and 7
Ligation 3 – ES3+7
Ligation 7 – ES4+6
Rxn Mixture – general
1x T4 Buffer
DNA
ddH2O
T4 Ligase .5uL/10uL rxn vol
10 or 20uL rxn vol

DNA  - NEB recommends 50ug vector and 1:3 vector:insert
Consensus OWW is 10ug and 6:1 vector:insert
OWWW says vector:insert is 1:1-6, all seem to work ok

OWW recommends 10uL rxn vol
NEB says 20uL

OWWà30min incubation at room temp
NEBà10min at same cond.

Aliquotted T4 Buffer into 100uL tubes (ATP is not stable)

Ligation 3 –
20uL rxn vol
2uL 10x T4 Buffer
1uL ES7D
1uL ES3 2hr Digest
1uL T4 Ligase
15uL H2O

1uL ES7 Digest
3uL ES3
13uL H2O

2uL ES7
4uL ES3
11uL H2O

Ligation 7 –
20uL rxn vol
2uL 10x T4 buffer
1uLES6
1uL ES4
15uL H2O
1uL T4 Ligase

1uL ES6
3uL ES4
13uL H2O

2uL ES6
4uLES4
11uL H2O

Incubate at room temp for 15min
Inactivate at 65 degrees Cfor 15 min

Transform using standard protocol (2uL DNA into 50uL comps with 20min incubation on ice with following controls
ddH2O –control
ddH2O+control
ES3 digest
ES4 Digest
ES6 Digest
ES7 Digest

Plated 35uL on LB amp plates

Phosphates ES6 digest
ES6 Digest UPL
2uL 10x T4 Buffer
2uL DNA
15uL H2O
1uL T4 Ligase
Incubate at room temp for 10 min
Inactivate at 65 degrees C for 10 min note setting ~1 on grey dry black gives 65 degrees C

Transformed using standard protocol
ES6 UPL (unphosphatased then ligated)
ES6 DL (phosphatased then ligated)
ES7 UPL
ES7 DL

Ligation results
Digested ES6 and ES7 give many transformants
Note that ES6 and ES7 had a bit more DNA than other transformations since we diluted other in ligation mix and neglected to dilute ES6 and ES7 pure digests to the same cone
Phosphatase appears to be working however. Ligated unphosphated ES6,7 gave many more transformants than phosphatased ligated ES6,7

Janet says this happen sometimes. Will screen 16 of each ligation.

Made 16 2mL cultures in LB Amp

Miniprep procedure as described in kit.

Restriction digests at minipreps.
Digesting all with EcoRI-HF+PstI
(36x)
5uL 10x NEB4à180uL
5uL10x BSAà180uL
.5uL EcoRI-HFà18uL
.5uLPstIà18uL
5uL DNA
34uL H2Oà1224uL

Restriction Digests placed in warm room
1.5 hour incubation
20 min inactivation at 80 degrees C

DàDigest
10uL loads of digest
5uL loads of undigested

ES9 Digest (also ran ES7)
5uL 10x NEB4
5uL 10x BSA
1uL EcoRI-HF
1uL PstI
5uL DNA
33uL H2O
Digest 1 hour at 37 degrees C
Kill 20min at 80 degrees C

Parts came
Streaked out onto Kan and Tet plates.

Streakouts worked
Note – tet plates dried up a bit (forgot to cover with big bin)

ESA and ESB are red as expected.
ES2 is purple – somewhat expected. Should not have promoter. Might be read-thru transcription)
Made 2mL liquid cultures in appropriate antibiotics.

Counting Colonies on L3 and L7 plates.
Name/#
Digested ES4 – 7
Digested ES6 – 512
L7 1:1 – 80
L7 2:4 – 247
Digest ES3 – 0
ES7 Ptest PL – 18
ES7 UPL – 204
ES6 PL Ptest – 22
L3 1:3 - 408
L3 1:3 – 218
Digested ES7 – 1280
+Control – 1280
-Control – 0
ES6 UPL – 194
L3 1:1 - 52
L3 2:4 – 600

Transformation with heat shock of RFP (it is in the PSBK3 backbone)

Thaw cells on ice
Aliquot 50uL
Add 2uL DNA
Incubate on ice 30min
Incubate at 42 degrees C
Incubate on ice for 2min
Add 500uL LB+IPTG
Incubate 1 hour at 37 degrees C on shaker (3hr)
Spread 100uL onto plates
Pellet leftover resuspend in 100uL and spread onto plates

Also received TrpR containing plasmid
Transformed using standard protocol
Plated 35uL, 15uL into liquid.

Backbones (calculations for ptrpL)
PSBIC3 50uL at 25ug/uL
We will digest ½ ugà20uL

Digest mix
5uL NEB4 10x
5uL BSA
1uL EcoRI-HF
1uL PstI
20uL DNA
18uL H2O

Incubate at 37 degrees C, kill at 80 degrees C

Oligo Hybridization
Rxn mix
3uL 100um sense oligo
3uL 100um antisense oligo
3uL PNK or T4 ligase buffer
3uL .5M NaCl
Put at 95 degrees C heat block for minutes kill power on heat block, allow to cool to room temperature

Ligation
Nucleotide ~660 daltons/pail
PSBIC3 is 2070ut=1370000g/mol
Digest had .5ug/50uL*mol/1370000g=7.3*10^-6 umol/uL

The hybridization should have 10uM=.01umol/uL
Want ~1:4 vector:insert
So 1uL vector~7.3*10^-6
We then want 4*7.3*10^-6 umol insert = 2.92*10^-5 which is .00292uL too small
Dilute insert to 1:100 for final conc of 1*10^-5 umol/L, will also try 1:10 dilution, then use 3uL
Ligation mix (L4)
20uL rxn volume
2uL 10x T4 buffer
1uL vector digest
3uL diluted insert
1uL T4 ligase
13uL H2O
10 minute room temp incubation
10 minute kill at 65 degrees Càthen standard transform

Plates from yesterday
RFP transformation
K-control conc 0
RFP conc ~150 (very small colonies)
K+control conc ~500
K-control 0
RFP 3 very small colonies
K+control ~300

TrpR transformation
Amp-Control 1
trpR 5 (2large with satellites,rest small)
Amp+control a lot

Ligation 4
40 colonies in all plates (including +control)
After continued incubation +control shows ~50 colonies, rest stil blank

Liquid cultures
Biobricks
ES2 no growth, restreaked from mail tube
ESA growth (brown)
ESB growth (red)
ES5 growth

trpR A+control growth
A-control no growth
trpR growth

will miniprep ESA, ESB, ES5, and trpR
Will retransform ligation 4 with heat shock and all

Digest of todays minipreps (4) will use EcoRI + PstI
50uL rxn vol
(x5)
5uL 10x NEB4à25uL
5uL10x NBSAà25uL
1uL EcoRi-HFà5uL
1uL PstIà5uL
10uL DNA
28uL ddH2Oà140uL

Transformation
Throw comps on ice
Aliquot 50uL
Add 2uL DNA
Incubate on ice 30in
Incubate at 42 degrees C 45 sec
Incubate on ice 2min
Add 500uL LB
Incubate 37 degrees C 1hr
Plate 300uL (not enough plates for spin down)

6/22
trpR-amp – streakout the big colonies and start new liquid culture
ESB – inducible Kan – looks fine
ES5 - inducible Kan- start liquid culture in LB-Kan-
ESA tet – start liquid culture

6/23
Transformation results –
L4
Ch1-control 0
Ch1+control 1000+
Annealed ptrpL 0
Digested pSBIC3 0
L4 1:10 1
L4 1:100 15 ß2 liquid cultures made

Es2 (re)streak looks good not dried out

Es2 liquids did not grow again, going to try multiple concentrations of tet

Stock 5mg/ml previous ones were 100x dilation=50ug/mL, going to try 50ug/mL, 25ug/mL, 12.5 ug/mL 6.25 ug/mL, 3.125 ug/mL

Digest
50uL volume for each of the 7 digersts
(x8)
5uL 10x buffer 4à40uL
5uL10x BSAà40uL
28uL H2Oà224uL
1uL enzyme1 EcoRIà8uL
1uL enzyme PstIà8uL

10uL DNA in each digest
trpR1, trpR2 RFP, ESA1, ESA2, ES5-1, ESA5-2

6/24
Miniprepped ES2(12.5x), 2xL4, RFP, ES5-1, ES5-2

TrpR Oligo Primers were received in the mail

Notes on previous nightsw cultures –
Both L4’s grew
ES2 100x did NOT grow
ES2 (12.5x) grew
Es2 50x did NOT
ES2 6.25x grew
ES2 25x did NOT
RFP grew
ES5-1 grew
ES5-2 grew

6/27
Digestion of MPS – RFP ES51, ES52, ES5, L4, L4, ES212.5
Master mix
500uL volume for each of the digests
(7x)
5uL 10x Buffer 4à35uL
5uL 10x BSAà35uL
33uL ddH2Oà231uL
1uL enzyme EcoRIà7uL
1uL enzyme PstIà7uL
10uL DNA of each MP

Notes –
L4 miniprep looks too large
Used filtered tip to accidentally to take up, soul mix was stuck lost 1/7th of mix
Added leftover mmix to weird L4

6/28
PCR L4
(2.2x)
5uL 10x thermopolà11uL
1uL 40mM dNTPà22uL
.5uL 100uM VF2à1.1uL
.5uL 100uM VRà 1.1uL
1uL plasmid
1uL ventà2.2uL
1uL MgSO4à2.2uL
40uL ddH2Oà 88uL

Miniprepped  trpR cultures (5mL)

Digest trpR MP with PstI
50uL rxn vol
5uL NEB4
5uL 10x BSA
1uL PstI
10uL DNA
29uL ddH2O

37 degrees C for 2 hours, 80 degrees C for 20 min

PCR ES5
(2.2x)
(2.2x)
5uL 10x thermopolà11uL
1uL 40mM dNTPà22uL
.5uL 100uM VF2à1.1uL
.5uL 100uM VRà 1.1uL
1uL plasmid
1uL ventà2.2uL
1uL MgSO4à2.2uL
40uL ddH2Oà 88uL

Digest of ES7
ES7 digested with XbaI, EcoRI
50uL rxn vol
5uL 10x NEB Buffer 4
5uL 10x BSA
1uL enzyme1
1uL enzyme2
5uL DNA
33uL ddH2O

2hr incubation at 37 degrees C, 20 min incubation at 80 degrees C
Took out 6uL saved in UPL tube
Added 5uL phosphatase buffer, 1uL phosphatase and incubated at 37 degrees C for one hr, killed at 80 degrees C for 20 min

L4 results still unclear. Going to digest L4 with just EcoRI and run one gel with digested pSBIC3. Hope to see size shift.
50uL rxn vol
5uL 10x NEB4
5uL10x BSA
1uL EcoRI-HF
5uL DNA
34uL ddH2O

37 degrees C for 1hr, 20 minutes at 80 degrees C

trpR PCR primers melting pt3 – forward 59.7 degrees C, reverse 47.1 degrees C
thermocycler settings
95 degrees C 2min
(30cycles of next 3)
95 degrees C 30sec
45 degrees C 30sec
74 degrees C 30sec
74 degrees C 5 min
4 degrees C hold

Rxn mix
50uL rxn vol
5uL 10x thermopol buffer
1uL 40mm dNTP
5uL 10uM F primer
5uL 10uM R primer
1uL plasmid
1uL MgSO4
1uL vent
31uL ddH2O

ES5 PCR digest
Just going to use ES5-1
50uL rxn vol
5uL 10x NEB4
5uL 10x BSA
1uL EcoRI-HF
1uL SpeI
1uL DynI
10uL PCR product
27uL ddH2O
1hr incubation at 37 degrees C, 20min incubation at 80 degrees C

Going to cut L4 with SpeI (both PCR and MP)
50uL rxn vol
(2.2x)
5uL 10x NEB4à11uL
5uL 10x BSAà11uL
1uL SpeIà2.2uL
5uL DNA
34uL H2Oà74.8uL

6/29
ES2 Testing
Cut eith XbaI, PstI,XbaI+PstI

XbaI, PstI
50uL rxn vol
5uL NEB4
5uL 10x BSA
1uL enzyme
5uL DNA
34uL water

xbaI+pstI
50uL rxn vol
5uL NEB4
5uL 10x BSA
1uL XbaI
1uL PstI
5uL DNA
33uL ddH2O

ES1 cut Eco Spe
50uL rxn vol
5uL NEB4
5uL 10x BSA
1uL EcoRI-HF
1uL SpeI
5uL DNA
33uL ddH2O

L6
20uL rxn vol
2uL 10x T4 ligase buffer
1uL T4 ligase
1/1/1/2uL vector
1/2/4/1uL insert
15/14/12/14uL ddH2O
Controls – phosphatased ES7, unphosphatased (3uL DNA,14uL H2O)
10 min room temp, 15 min at 80 degrees C, transformed using standard procedure.

Ran ES7+ES5 digest controls separately

trpR PCR product digestion
going to digest with EcoRI PstI and DpnI
50uL rxn mix
5uL 10x NEB4
5uL 10x BSA
1uL EcoRI-HF
1uL PstI
1uL DpnI
10uL DNA
27uL H2O

pSBIA3 (linear) digestion
50uL rxn vol
5uL 10x NEB4
5uL 10x BSA
1uL EcoRI-HF
1uLPstI
1uL DpnI
20uL DNA
17uL ddH2O

L9 (not in ppt yet) and trpR ligation L10 (trpRàpSBIA3)

20uL rxn volume
2uL 10x T4 Ligase buffer
1uL T4 Ligase
1uL vector
1uL insert
15uL ddH2O

ES1-insert
ES6- vector
Transformed using standard protocol

6/30
Transformation results –
Control+ - 488
ES1 – 164
+Control  - A lot
ES7 UPL – 109 w/ satellites
ES7 PL – 17+10satellites
L6 1:1 – 20
L6 2:1 – 10
ES7 – 30
L6 1:2 – 12+2satellites
L6 1:4 – 7+1satellite
L9 1:1 – 35
L9 1:3 – 264
YSB143 Digest – 7
ES7 Digest – 8
ES5 Digest – 8
ES5 Digest – 0
L TrpR 1:1 – 2
-control – 0
LtrpR 1:3 – 3
-control 0
-control 2
+control – a lot+many satellites

L4 restriction Test – 50uL rxn vol
(3.3x)
5uL 10x NEB4à16.5uL
5uL10x BSAà16.5uL
1uL SpeIà3.3uL
10uL DNA
29uL ddH2Oà95.7uL

7/1
L6 1:1 and L9 1:1 removed from incubator and put in fridge

Standard minprep procedure used.
LtrpR –A-F all had growth present.

Miniprepped DNA stored in freezer.

7/28
PCR L81
50uL rxn vol
25uL 2x phusion mmix
2.5uL VF2 primer
2.5uL VR primer
1uL DNA
19uL ddH2O

8/15
PCR ES9, T1, L11, trpR
50uL rxn vol
25uL 2x phusion
2.5uL VF2 primer
2.5uL VR primer
1uL DNA
19uL H2O

ES1, L5, L8
50uL rxn vol
5uL NEB4
5uL 10x BSA
5uL DNA
33uLddH2O
1uL enzyme 1
1uL enzyme2

L11 trpR, T1, ES9 PCRs digestion
5uL NEB4
3uL 10x BSA
10uL DNA
1uL DpnI
1uL enzyme 1
1uL enzyme2
27uL H2O

Made glycerol stocks of CFP, GFP, minipreppred CFP, GFP, retransformed L11 using standard protocol.

8/16
Redo L5 and L8 digestions.

Phosphatase L5 and L8
50uL rxn vol
5uL phos. Buffer
1uL phosphatase
44uL DNA

Ligation L12 and L17
20uL rxn vol
2uL T4 ligase buffer
1uL backbone
3uL insert
1uL T4 Ligase
13uL H2O

Transformed L17,L12

Ligation trpR in pSBIC3
20uL rxn vol
2uL T4 ligase buffer
5uL backbone
3uL insert
9uL H2O
1uL ligase

Transformed L19

8/17
Miniprepped L11 A-B

Plac, L18 digestion
50rxn vol
5uL NEB4
5uL 10x BSA
5uL DNA
33uL ddH2O
1uL enzyme1
1uL enzyme2

Transformation results –
L8 DSP – 2
L11 DDXP – 0
L17 – 2
L5 DSP – 2
L5 DSP – 19
+Amp – 0
T1 DDXP – 0
-amp – 0
L12 – 0
trpR ch1 – 0
pSBIC3 – 0
trpR digest – 0

cultured 2 L17 colonies

8/22
Zipper PCR
50uL rxn vol
2.5 uL F primer
2.5uL R primer
1uL DNA
25uL phusion
19uL H2O

L12 test digest 5 and one ES1 digest for L19
50uL rxn vol
5uL 10x NEB4
5uL 10x BSA
1uL enzyme1
1uL enzyme 2
10uL enzyme
28uL ddH2O

8/23
PCR GFP
50 rxn vol
25uL phusion
2.5uL GFP MF
2.5uL MR
1uL 19uL

Ligations trpR+pSBIC3, L19, ES9+ES1, L4+L18, L11+plac
20uL rxn vol
2uL T4 ligase buffer
1uL backbone
3uL insert
1uL T4 Ligase
13uL dH2O

Ligations –
L19 vector ES1, Insert ES9
710 – insert 710nzipper, vector psBIC3
711 – insert 711czippe, vector pSBIC3

8/24
Transformations
L19 – 4-5
L4+L18 – 3
+amp ES7/+Amp – a lot
pLac+L11 – 0
-control (all) – 0
710
711
+ahl?

L3 and T1 digestions –
L3 and (DES)
50rxn vol
5uL NEB4
5uL 10x BSA
10uL DNA
1uL DpnI
1uL EcoRI
1uL SpeI
27uL ddH2O

T1
50uL rxn vol
5uL NEB4
5uL 10x BSA
5uL DNA
1uL EcoRI
1uL XbaI
33uL ddH2O

L22 Ligations (L3+T1)
20uL rxn vol
2uL T4 Ligase buffer
1uL backbone
3uL insert
1uL T4 ligase
13uL ddH2O

Miniprepped L4+L18, L19A,B

8/25
Note – L4+18 cultures are noticeably red. (good)
Just going to send everything for sequencing. Not much to gain from gel.

Started cultures of L12A, trpR pSBIC3, L12A, L710A-B, L711A-B, L3-3, plae+L11AB, trpRC

Miniprepped cultures
Test cats –
(11x)
25uL rxn vol
2.5uL 10x NEB4à27.5uL
2.5uL 10x BSAà27.5uL
1uL EcoRI-HFà11uL
1uL PstIà 11uL
5uL DNA
13uL ddH2Oà143uL

8/30
GFP round the horn
Mutagenesis ligation
20uL rxn vol
2uL 10x T4 ligase buffer
1uL T4 ligase
2uL DNA
15uL ddH2O

L3-3 digestion
50uL rxn vol
5uL NEB4
5uL 10x BSA
5uL DNA
1uL SpeI
1uL PstI
33uL ddH2O

L22 Ligation
20uL rxn vol
2uL 10x T4 ligase buffer
1uL backbone L3
3uL insert T1
1uL ligase
13uL ddH2O

Cultured L19A-C, L22, transformed L22, GFP

8/31
GFP/L22 transformations
-control – 0
GFP – a lot
L22 – 42

Miniprepped L22B,C, arbitrarily did not miniprep A, put in fridge.

9/8
OBS – subculture of DH3OC comp cells wuth different concs of parent culture
500L/5mL
1000L/5mL

Incubation time – 17hrs

Aronch period should be in the log phase to dilute stock to 1:100. Diluted stock should be grown from morning.

9/12
711 pCR CZ
50uL rxn vol
25uL phusion
2.5uL F primer
2.5uL R primer
1uL DNA
19uL dH2O

GFP miPCR
50uL rxn vol
25uL phusion
2.5uL GFP MF
2.5uL GFP MR
1uL DNA GFPmi
19uL ddH2O

L3-3 PCR
25uL phusion
2.5 VF2
2.5uL VR
1uL DNA
19uL ddH2O

41PCR
25uL phusion
2.5uL VF2
2.5uL VR
1uL DNA
19uL ddH2O

GFPlm PCR
25uL phusion
2.5uL VF2
2.5uL VR
1uL DNA
19uL ddH2O

L3, L11 PCRs purified

L3, L11 digests (going to gel extract so DpnI unnecessary)
L3 – EcoRI-HF, SpeI
L11 0 XbaI, PstI
5uL 10x NEB4
5uL 10x BSA
1uL enzyme 1
1uL enzyme 2
20uL DNAßsince we are gel extracting
18uL ddH2O

8/13
GFPml, PCR, 711 PCR DD purified
711 PCR DD purified with EcoRI-HF, PstI (to ligate into pSBIC3)
5uL 10x NEB4
5uL 10x BSA
1uL Fzce
1uL Pst
10uL DNA
28uL ddH2O

L3,L11 digests killed at 80 degrees C
ES4-ES1 sent for sequencing. (all of today’s tubes put in green rack)
Miniprepped ES1
711 digest killed at 80 degrees C

Comp cellsàsee protocol on OWW
Tfse of comp cells

Place on amp is used as a test
Black mark is second half of tube that was repelleted, may effect competency
Amp placA+Amp placB
LB plNCA+NCB=negative control

Ligations
L22 insert L3
Backbone T1
20uL rxn vol
2uL 10x T4 buffer
1uL backbone
3uL insert
1uL T4 ligase
13uL ddH2O

Plate L11, insert L11, backbone plae

Transformed with janet’s cells using standard protocols

9/14
Started 2 cultures each of 711, L22
Streaked out (poorly)plae+L11

9/15
Miniprepped 711, L22 cultures
Restreaked plae+L11

GFP nzipped digests
GFPmiPCR
50uL rxn vol
5uL 10x NEB4
5uL 10x BSA
1uL AgeI-HF
1uL EcoRI-HF
10uL DNA
1uL DpnI
27uL ddH2O

NZipper
50uL rxn vol
5uL 10x NEB4
5uL 10x BSA
1uL EcoRI-HF
1uL NgoMIV
10uL DNA
28uL ddH2O

GFPml Nzipper ligation (NZGFP)
Insert GFPml
Backbone 710A
20uL rxn vol
2uL 10x T4 ligase buffer
1uL 710A
3uL GFPml
1uL T4 ligase
13uL ddH2O

Restreaked plae+L11

Transformed NZGFP using quick protocol

L12 Digestion
50uL rxn vol
5uL 10x NEB4
5uL 10x BSA
1uL EcoRI-HF
1uL XbaI
10uL DNA
28uL ddH2O

Order ES1 and retransform ES1
Send ES9 sequences to Vershon
Redesign lovtap mutagenesis primers
Write up adder experimental part

Miniprep plac+L11

PCR plac+L11A
25uL phusion
2.5uL VF2
2.5uL VR
1uL DNA
19uL ddH2O

PCR purify L22 PCR and L12ADEX

L22 Digestion DDES
5uL NEB4
5uL 10x BSA
10uL DNA
1uL DPN1
1uL EcoRI
1uL SpeI
27 ddH2O

L4+L18 digestion DEX
5uL NEB4
5uL 10x BSA
1uL EcoRI
1uL XbaI
10uL DNA
28uL ddHwO

PCR purify L4+L18 and plac+L11 PCR

Plac+L11 digestion DDES
5uL NEB4
5uL 10x BSA
1uL EcoRI
1uL SpeI
10uL DNA
1uL DPNI
27uL ddH2O

Ligate L22+L12
2uL 10x T4 ligase buffer
1uL backbone L12
3uL insert L22
1uL ligase
13uL ddH2O

L21 transformation/colonies
-control – 0
+control – a lot
L21 – 0
L22 DDES – 0
L12 DEX – 5

9/19
Miniprepped ES1A,B
Culture plac/L11+L4/L18A,B, L21A-B

9/20
Miniprep all cultures made on 9/19

9/22
NZGFP ligation
20uL rxn vol
2uL 10x T4 ligase buffer
3uL 710A
9uL GFPml
1uL T4 ligase
5uL ddH2O

L21
Backbone L12DEX
Insert L21 DDES
(S/T)
20/20uL rxn vol
2/2uL T4 buffer
1/2uL backbone
6/2uL insert
1/1uL T4 ligase
10/13uL ddH2O

Started new cultures of ESB, RBS lovtap term. Promoter RBS lovtap term.

9/23
Miniprepped cultures from 9/22