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Team:Chiba/Notebook 1
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2010-08-31
Making plate with BrothAmp x 13 Kan x 1 Amp + IPTG x 1
Transformation
R0010 B0030 B0032 B0033 K145270 I746903 R0051 C0051 K145001 C0012 K145001 C0012 P0312 I719015
2010-09-02
Mini-prep(DNA transformated in August 31th)I13522 C0040 C0052 C0053 C0080 C0072 I0500 Q04510 Q04400 Q04121 R0040 B0014 B1101 I715038 K081012 K082034 K113007 I763011
2010-09-03
Preparing for experimentWe inserted plasmid with cI promoter + GFP to E.coli. And
2010-09-04
Preparing for experimentI746903
2010-09-05
K091107 R0065 K145150 Q01121 Q01511 Q03121 Q03400 Q03530 Q04511 J06800 J06801
2010-09-06
Co-transformationPlux – gfp + Pc each 1uL SOC 200uL
2010-09-07
E0240 R0061 I0500 C0080 C0072 Q04510 Q04400 Q04121
2010-09-08
TransformationXL10GkanR 30uL DNA 1uL SOC 100uL K145269 K145205 R1062 I0462 P0440 K091204 J01101 P0140 K091204 J01101 P0140 P0340 B0034
2010-09-14
1) K091204(2-8J) Pc – luxR function check [Pc – luxR(pSB1A2) + Plux – GFP(pAC)] each 1uL + XL10GkanR 50uL AHL : 30C8HSL(1/10000 of 1μM) 2) T9002
2010-09-16
PCR
T7/cI(OR1/2) – GFP
(1) (2)
--------------------------------
template 1uL
primer 5uL
F 5uL
R 5uL
10x buffer 5uL
dNTP 5uL
NFW 28uL
ventP 1uL
-------------------------------
50uL 50uL
Transformation of BBa_J04450(3A) -> incubation
2010-09-19
From 2010 Biobrick plasmid with Cm antibiotic ------------- 1-3C 1-3A pSB1C3 plasmid with Amp antibiotic ------------- 1-1K pSB1A3 ↓ To transformate total five types, we seeded to plates. ↓ PCR products ↓ Gel extraction ↓ ← ADB buffer ZYMO purification
1. To prepare 2ml tube(for ZYMO purification) + column, add the solution and operate centrifuge in 1 minute. Throw away left solution.
2. Same to mini-prep
* wash buffer 200uL -> centrifuge 30sec -> throwing away left solution. X2 * wash buffer 200uL -> centrifuge 1min -> throwing away left solution. X1 * operating centrifuge in 1min with empty tube -> throwing away left solution. X1
3. NFW elution(10uL) Add NFW and wait 1min. Operate centrifuge in 1min 4. Gel electrophoresis(each 1uL)
SOEing PCR ①T7/cI(OR2)-F-R ②T7/cI(OR1)-F-R ③Ptet-luxR-(OR2) ④Ptet-luxR-(OR1) ⑤Prom-luxR-(OR2) ⑥Prom-luxR-(OR1) 1. ①1uL, ③3uL 2. ②1uL, ④4uL 3. ①1uL, ⑤1uL 4. ②1uL, ⑥1uL
1. ①-③
2. ②-④
--------------------------
template 1uL-4uL
primer Fwd -
primer Rev -
dNTP 5uL
10xbuffer 5uL
NFW 34uL
VentP 1uL
-------------------------
50uL
3. ①-⑤
4. ②-⑥
-------------------------
template 1uL-1uL
primer Fwd -
primer Rev -
dNTP 5uL
10xbuffer 5uL
NFW 37uL
VentP 1uL
-------------------------
50uL
5. control
-------------------------
template 1uL
primer Fwd 5uL
primer Rev 5uL
dNTP 5uL
10xbuffer 5uL
NFW 28uL
VentP 1uL
-------------------------
50uL
PCR
94°C – 5min
94°C – 15sec --
52°C – 30sec │- 10cycles
72°C – 1min --
72°C – 10min
----------------
4°C stop
2010-09-20
TransformationBBa_P0453(B0034-C2P22-T-T) RBS_C2P22 for PCR template 2-1 P pSB1AK3 11:30 ~ DNA : 1uL XL10GkanR : 30uL SOC : 100uL
Cm Amp 1-3C 1-1K 1-3A pSB1A3 pSB1C3
liquid incubation(37°C) 7:15~ Mini-prep
ZYMO purification
From PCR product from SOEing PCR (09-19) 1. Add PCR products(50uL) and DNA binding buffer(200uL) into 1.5mL tube(Commonly adding DNA binding buffer for double amount of PCR products, but it must be 200uL at least). After that, mix this tube for a second with vortex and move to 2mL tube with column. After operating centrifuge, transfer remaining a little PCR product to 2mL tube with column. 2. centrifuge 1min → throwing away left solution 3. Same to mini-prep * wash buffer 200uL -> centrifuge 30sec -> throwing away left solution. X2 * wash buffer 200uL -> centrifuge 1min -> throwing away left solution. X1 * operating centrifuge in 1min with empty tube -> throwing away left solution. X1 4. NFW elution(10uL)
↓
PCR
PCR Condition 94°C – 5min 94°C – 15sec -- 52°C – 30sec │-10 cycles 72°C – 1min -- 72°C – 10min ---------------- 4°C stop
After 10 cycles, we added primer to SOEing PCR product(completed ZYMO purification) ------------------------- template 10uL primer Fwd 5uL primer Rev 5uL dNTP 5uL 10xbuffer 5uL NFW 19uL VentP 1uL ------------------------- 50uL
Using primer 1. ①-③ Fwd : Ptet-luxR∙FA-R Rev : TcIg∙FA-R 2. ②-④ Fwd : Ptet-luxR∙FA-R Rev : TcIg∙FA-R 3. ①-⑤ Fwd : PluxR∙FA-R Rev : TcIg∙FA-R 4. ②-⑥ Fwd : PluxR∙FA-R Rev : TcIg∙FA-R 5. control
Using template 1. Ptet-LuxR-PT7/cI(OR2)-GFP 2. Ptet-LuxR-PT7/cI(OR1)-GFP 3. Prom-LuxR-Pt7/cI(OR2)-GFP 4. Prom-LuxR-Pt7.cI(OR1)-GFP 5. control
PCR condition(usingTAKARA PCR thermal cycler) 94°C – 5min 94°C – 15sec -- 51°C – 30sec │-25 cycles 72°C – 1min -- 72°C – 10min
↓
Gel Electrophoresis
Cm Amp 1-3C 1-1K 1-3A pSB1A3 pSB1C3
↓
Liquid Incubation(37°C) 7:15~
-------------------------
template 3uL
primer Fwd 5uL
primer Rev 5uL
dNTP 5uL
10xbuffer 5uL
NFW 26uL
VentP 1uL
-------------------------
50uL
Using primer
Fwd : pSB1C3FA-F
Rev : pSB1C3FA-R
Using template
1. 1-3C(J04450,pSB3C5)
2. pSB1C3
3. 1-3A(J04450,pSB1C3)
4. 1-1K(J04450,J63010)
5. control(pCI-GFP) primer Fwd : PcI-GFP∙fwd Rev : PcI-GFP∙rev
PCR condition
94°C – 5min
94°C – 15sec --
51°C – 30sec │-25 cycles
72°C – 1min --
72°C – 10min
↓
Gel Electrophoresis
Notebook
8/31 We've started experiment. First, we researched the property of T7 promoter.
9/5 We are researching promoter and inverters.
10/7 We are on the bench!! Design primers, PCR and experimentsssssssssss :D
