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Team:Chiba/Notebook 1
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Revision as of 14:40, 21 October 2010
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 1㎕ SOC 200㎕
2010-09-07
E0240 R0061 I0500 C0080 C0072 Q04510 Q04400 Q04121
2010-09-08
TransformationXL10GkanR 30㎕ DNA 1㎕ SOC 100㎕ 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 1㎕ + XL10GkanR 50㎕ AHL : 30C8HSL(1/10000 of 1μM) 2) T9002
2010-09-16
PCR
T7/cI(OR1/2) – GFP
(1) (2)
--------------------------------
template 1㎕
primer 5㎕
F 5㎕
R 5㎕
10x buffer 5㎕
dNTP 5㎕
NFW 28㎕
ventP 1㎕
-------------------------------
50㎕ 50㎕
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 200㎕ -> centrifuge 30sec -> throwing away left solution. X2 * wash buffer 200㎕ -> centrifuge 1min -> throwing away left solution. X1 * operating centrifuge in 1min with empty tube -> throwing away left solution. X1
3. NFW elution(10㎕) Add NFW and wait 1min. Operate centrifuge in 1min 4. Gel electrophoresis(each 1㎕)
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. ①1㎕, ③3㎕ 2. ②1㎕, ④4㎕ 3. ①1㎕, ⑤1㎕ 4. ②1㎕, ⑥1㎕
1. ①-③
2. ②-④
--------------------------
template 1㎕-4㎕
primer Fwd -
primer Rev -
dNTP 5㎕
10xbuffer 5㎕
NFW 34㎕
VentP 1㎕
-------------------------
50㎕
3. ①-⑤
4. ②-⑥
-------------------------
template 1㎕-1㎕
primer Fwd -
primer Rev -
dNTP 5㎕
10xbuffer 5㎕
NFW 37㎕
VentP 1㎕
-------------------------
50㎕
5. control
-------------------------
template 1㎕
primer Fwd 5㎕
primer Rev 5㎕
dNTP 5㎕
10xbuffer 5㎕
NFW 28㎕
VentP 1㎕
-------------------------
50㎕
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 : 1㎕ XL10GkanR : 30㎕ SOC : 100㎕
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(50㎕) and DNA binding buffer(200㎕) into 1.5mL tube(Commonly adding DNA binding buffer for double amount of PCR products, but it must be 200㎕ 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 200㎕ -> centrifuge 30sec -> throwing away left solution. X2 * wash buffer 200㎕ -> centrifuge 1min -> throwing away left solution. X1 * operating centrifuge in 1min with empty tube -> throwing away left solution. X1 4. NFW elution(10㎕)
↓
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 10㎕ primer Fwd 5㎕ primer Rev 5㎕ dNTP 5㎕ 10xbuffer 5㎕ NFW 19㎕ VentP 1㎕ ------------------------- 50㎕
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 3㎕
primer Fwd 5㎕
primer Rev 5㎕
dNTP 5㎕
10xbuffer 5㎕
NFW 26㎕
VentP 1㎕
-------------------------
50㎕
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
