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From 2010.igem.org

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