Team:Chiba/Notebook 1

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2010-08-31

Making plate with Broth

Amp 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 experiment

We inserted plasmid with cI promoter + GFP to E.coli. And 

2010-09-04

Preparing for experiment

I746903

2010-09-05

K091107 R0065 K145150 Q01121 Q01511 Q03121 Q03400 Q03530 Q04511 J06800 J06801

2010-09-06

Co-transformation

Plux – gfp + Pc each 1㎕
SOC 200㎕

2010-09-07

E0240 R0061 I0500 C0080 C0072 Q04510 Q04400 Q04121

2010-09-08

Transformation

XL10GkanR 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

Transformation

BBa_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