Team:Chiba/Notebook 1

From 2010.igem.org

(Difference between revisions)
Line 210: Line 210:
<html><h3>2010-09-06</h3></html>
<html><h3>2010-09-06</h3></html>
Co-transformation
Co-transformation
-
  Plux – gfp + Pc each 1㎕
+
  Plux – gfp + Pc each 1uL
-
  SOC 200㎕
+
  SOC 200uL
<html><h3>2010-09-07</h3></html>
<html><h3>2010-09-07</h3></html>
Line 218: Line 218:
<html><h3>2010-09-08</h3></html>
<html><h3>2010-09-08</h3></html>
Transformation
Transformation
-
  XL10GkanR 30㎕ DNA 1㎕ SOC 100㎕
+
  XL10GkanR 30uL DNA 1uL SOC 100uL
  K145269 K145205 R1062 I0462 P0440 K091204 J01101 P0140 K091204 J01101 P0140 P0340 B0034
  K145269 K145205 R1062 I0462 P0440 K091204 J01101 P0140 K091204 J01101 P0140 P0340 B0034
<html><h3>2010-09-14</h3></html>
<html><h3>2010-09-14</h3></html>
  1) K091204(2-8J) Pc – luxR function check
  1) K091204(2-8J) Pc – luxR function check
-
   [Pc – luxR(pSB1A2) + Plux – GFP(pAC)] each 1㎕ + XL10GkanR 50㎕
+
   [Pc – luxR(pSB1A2) + Plux – GFP(pAC)] each 1uL + XL10GkanR 50uL
   AHL : 30C8HSL(1/10000 of 1μM)
   AHL : 30C8HSL(1/10000 of 1μM)
  2) T9002  
  2) T9002  
Line 232: Line 232:
               (1)      (2)
               (1)      (2)
   --------------------------------
   --------------------------------
-
     template  1㎕
+
     template  1uL
-
     primer    5㎕
+
     primer    5uL
-
     F          5㎕
+
     F          5uL
-
     R          5㎕
+
     R          5uL
-
     10x buffer 5㎕
+
     10x buffer 5uL
-
     dNTP      5㎕
+
     dNTP      5uL
-
     NFW        28㎕
+
     NFW        28uL
-
     ventP      1㎕
+
     ventP      1uL
   -------------------------------
   -------------------------------
-
               50㎕     50㎕
+
               50uL     50uL
   Transformation of BBa_J04450(3A) -> incubation
   Transformation of BBa_J04450(3A) -> incubation
Line 262: Line 262:
2. Same to mini-prep
2. Same to mini-prep
-
   * wash buffer 200㎕ -> centrifuge 30sec -> throwing away left solution.  X2
+
   * wash buffer 200uL -> centrifuge 30sec -> throwing away left solution.  X2
-
   * wash buffer 200㎕ -> centrifuge 1min -> throwing away left solution.  X1
+
   * wash buffer 200uL -> centrifuge 1min -> throwing away left solution.  X1
   * operating centrifuge in 1min with empty tube -> throwing away left solution.  X1
   * operating centrifuge in 1min with empty tube -> throwing away left solution.  X1
-
3. NFW elution(10㎕)
+
3. NFW elution(10uL)
Add NFW and wait 1min. Operate centrifuge in 1min
Add NFW and wait 1min. Operate centrifuge in 1min
-
4. Gel electrophoresis(each 1㎕)
+
4. Gel electrophoresis(each 1uL)
  SOEing PCR
  SOEing PCR
Line 276: Line 276:
   ⑤Prom-luxR-(OR2)
   ⑤Prom-luxR-(OR2)
   ⑥Prom-luxR-(OR1)
   ⑥Prom-luxR-(OR1)
-
  1. ①1㎕, ③3㎕
+
  1. ①1uL, ③3uL
-
  2. ②1㎕, ④4㎕
+
  2. ②1uL, ④4uL
-
  3. ①1㎕, ⑤1㎕
+
  3. ①1uL, ⑤1uL
-
  4. ②1㎕, ⑥1㎕
+
  4. ②1uL, ⑥1uL
  1. ①-③
  1. ①-③
  2. ②-④
  2. ②-④
   --------------------------
   --------------------------
-
   template    1㎕-4㎕
+
   template    1uL-4uL
   primer Fwd  -
   primer Fwd  -
   primer Rev  -
   primer Rev  -
-
   dNTP        5㎕
+
   dNTP        5uL
-
   10xbuffer  5㎕
+
   10xbuffer  5uL
-
   NFW        34㎕
+
   NFW        34uL
-
   VentP      1㎕
+
   VentP      1uL
   -------------------------
   -------------------------
-
               50㎕<br>
+
               50uL<br>
  3. ①-⑤
  3. ①-⑤
  4. ②-⑥
  4. ②-⑥
   -------------------------
   -------------------------
-
   template      1㎕-1㎕
+
   template      1uL-1uL
   primer Fwd    -
   primer Fwd    -
   primer Rev    -
   primer Rev    -
-
   dNTP          5㎕
+
   dNTP          5uL
-
   10xbuffer    5㎕
+
   10xbuffer    5uL
-
   NFW          37㎕
+
   NFW          37uL
-
   VentP        1㎕
+
   VentP        1uL
   -------------------------
   -------------------------
-
                 50㎕<br>
+
                 50uL<br>
  5. control
  5. control
   -------------------------
   -------------------------
-
   template      1㎕
+
   template      1uL
-
   primer Fwd    5㎕
+
   primer Fwd    5uL
-
   primer Rev    5㎕
+
   primer Rev    5uL
-
   dNTP          5㎕
+
   dNTP          5uL
-
   10xbuffer    5㎕
+
   10xbuffer    5uL
-
   NFW          28㎕
+
   NFW          28uL
-
   VentP        1㎕
+
   VentP        1uL
   -------------------------
   -------------------------
-
                 50㎕<br>
+
                 50uL<br>
  PCR
  PCR
   94°C – 5min
   94°C – 5min
Line 329: Line 329:
  BBa_P0453(B0034-C2P22-T-T) RBS_C2P22 for PCR template
  BBa_P0453(B0034-C2P22-T-T) RBS_C2P22 for PCR template
  2-1 P    pSB1AK3
  2-1 P    pSB1AK3
-
  11:30 ~  DNA : 1㎕   XL10GkanR : 30㎕   SOC : 100㎕<br>
+
  11:30 ~  DNA : 1uL   XL10GkanR : 30uL   SOC : 100uL<br>
  Cm              Amp
  Cm              Amp
  1-3C            1-1K
  1-3C            1-1K
Line 338: Line 338:
ZYMO purification
ZYMO purification
  From PCR product from SOEing PCR (09-19)
  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
+
  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 200㎕ at least). After that, mix this tube for a second with vortex and move
+
  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.
  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
  2. centrifuge 1min → throwing away left solution
  3. Same to mini-prep
  3. Same to mini-prep
-
   * wash buffer 200㎕ -> centrifuge 30sec -> throwing away left solution.  X2
+
   * wash buffer 200uL -> centrifuge 30sec -> throwing away left solution.  X2
-
   * wash buffer 200㎕ -> centrifuge 1min -> throwing away left solution.  X1
+
   * wash buffer 200uL -> centrifuge 1min -> throwing away left solution.  X1
   * operating centrifuge in 1min with empty tube -> throwing away left solution.  X1
   * operating centrifuge in 1min with empty tube -> throwing away left solution.  X1
-
  4. NFW elution(10㎕)
+
  4. NFW elution(10uL)
↓<br>
↓<br>
PCR
PCR
Line 359: Line 359:
  After 10 cycles, we added primer to SOEing PCR product(completed ZYMO purification)
  After 10 cycles, we added primer to SOEing PCR product(completed ZYMO purification)
   -------------------------
   -------------------------
-
   template      10㎕
+
   template      10uL
-
   primer Fwd    5㎕
+
   primer Fwd    5uL
-
   primer Rev    5㎕
+
   primer Rev    5uL
-
   dNTP          5㎕
+
   dNTP          5uL
-
   10xbuffer      5㎕
+
   10xbuffer      5uL
-
   NFW            19㎕
+
   NFW            19uL
-
   VentP          1㎕
+
   VentP          1uL
   -------------------------
   -------------------------
-
                 50㎕<br>
+
                 50uL<br>
  Using primer
  Using primer
   1. ①-③ Fwd : Ptet-luxR∙FA-R  Rev : TcIg∙FA-R
   1. ①-③ Fwd : Ptet-luxR∙FA-R  Rev : TcIg∙FA-R
Line 395: Line 395:
Liquid Incubation(37°C) 7:15~
Liquid Incubation(37°C) 7:15~
  -------------------------
  -------------------------
-
  template      3㎕
+
  template      3uL
-
  primer Fwd    5㎕
+
  primer Fwd    5uL
-
  primer Rev    5㎕
+
  primer Rev    5uL
-
  dNTP          5㎕
+
  dNTP          5uL
-
  10xbuffer      5㎕
+
  10xbuffer      5uL
-
  NFW            26㎕
+
  NFW            26uL
-
  VentP          1㎕
+
  VentP          1uL
  -------------------------
  -------------------------
-
                 50㎕<br>
+
                 50uL<br>
  Using primer
  Using primer
   Fwd : pSB1C3FA-F
   Fwd : pSB1C3FA-F

Revision as of 15:56, 26 October 2010




 




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 1uL
SOC 200uL

2010-09-07

E0240 R0061 I0500 C0080 C0072 Q04510 Q04400 Q04121

2010-09-08

Transformation 

XL10GkanR 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

Transformation 

BBa_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
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