Team:Chiba/Notebook 1

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                <title>Free CSS Navigation Menu Designs 2 at exploding-boy.com</title>
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                                        <!-- CSS Tabs -->
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<li><a href="https://2010.igem.org/Team:Chiba"><span>Home</span></a></li>
<li><a href="https://2010.igem.org/Team:Chiba"><span>Home</span></a></li>
-
<li><a href="https://2010.igem.org/Team:Chiba/Team"><span>Team</span></a>
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<li><a href="https://2010.igem.org/Team:Chiba/Team"><span>Team</span></a></li>
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<ul style="z-index:1"> <br>
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<li><a href="https://2010.igem.org/Team:Chiba/Project"><span>Project</span></a></li>
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<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Team">Team</a></li> <li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Member">Member</a></li>
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<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/School">School</a></li>
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</ul>
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</li>
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<li><a href="https://2010.igem.org/Team:Chiba/Project"><span>Project</span></a>
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<ul style="z-index:1"><br>
+
-
                        <li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Project">Overall Project</a></li>
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-
<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Circuit_1">Circuit 1</a></li>
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<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Circuit_2">Circuit 2</a></li>
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-
</ul>
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</li>
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<li><a href="https://2010.igem.org/Team:Chiba/Parts"><span>Parts</span></a></li>
<li><a href="https://2010.igem.org/Team:Chiba/Parts"><span>Parts</span></a></li>
-
<li><a href="https://2010.igem.org/Team:Chiba/Modeling"><span>Modeling</span></a></li>
+
<li><a href="https://2010.igem.org/Team:Chiba/Notebook"><span>Notebook</span></a></li>
-
<li><a href="https://2010.igem.org/Team:Chiba/Result"><span>Result</span></a>
+
-
<ul style="z-index:1"><br>
+
-
                        <li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Plasmid1">Plasmid 1</a></li>
+
-
<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/Plasmid2">Plasmid 2</a></li>
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-
<li><a class="tablinks" href="https://2010.igem.org/Team:Chiba/lux_promoter">lux Promoter</a></li><br>
+
-
</ul>
+
-
</li>
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-
<li id="current"><a href="https://2010.igem.org/Team:Chiba/Notebook"><span>Notebook</span></a></li>
+
<li><a href="https://2010.igem.org/Team:Chiba/Support"><span>Support</span></a></li>
<li><a href="https://2010.igem.org/Team:Chiba/Support"><span>Support</span></a></li>
 +
<li id="current"><a href="https://2010.igem.org/Team:Chiba/Safety"><span>Safety</span></a></li>
-
                                </ul>
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 +
                <ul>
 +
                                <!-- CSS Tabs -->
 +
<li><a href="https://2010.igem.org/Team:Chiba/Notebook"><span>Notebook 1</span></a></li>
 +
<li><a href="https://2010.igem.org/Team:Chiba/Notebook_2"><span>Notebook 2</span></a></li>
 +
<li><a href="https://2010.igem.org/Team:Chiba/Notebook_3"><span>Notebook 3</span></a></li>
 +
                        </ul>
 +
                </div>
 +
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 +
<!--- 2nd tab End--->
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<!--- Background Contents End --->
<!--- Main Contents Start --->
<!--- Main Contents Start --->
-
<br><br>
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<br><br><br>
<html><h3>2010-08-31</h3></html>
<html><h3>2010-08-31</h3></html>
-
<Making plate with Broth>
+
Making plate with Broth
  Amp x 13  
  Amp x 13  
  Kan x 1  
  Kan x 1  
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<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>
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<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  
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               (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
<html><h3>2010-09-19</h3></html>
<html><h3>2010-09-19</h3></html>
-
  From 2010 Biobrick
+
From 2010 Biobrick
-
  plasmid with Cm antibiotic  -------------
+
  plasmid with Cm antibiotic  -------------
-
    1-3C 1-3A pSB1C3
+
  1-3C 1-3A pSB1C3
-
  plasmid with Amp antibiotic  -------------  
+
  plasmid with Amp antibiotic  -------------  
-
    1-1K pSB1A3
+
  1-1K pSB1A3
-
↓
+
  ↓
-
To transformate total five types, we seeded to plates.
+
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.
-
 
-
 
-
 
-
 
-
 
-
 
-
  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
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
-
①T7/cI(OR2)-F-R
+
  ①T7/cI(OR2)-F-R
-
②T7/cI(OR1)-F-R
+
  ②T7/cI(OR1)-F-R
-
③Ptet-luxR-(OR2)
+
  ③Ptet-luxR-(OR2)
-
④Ptet-luxR-(OR1)
+
  ④Ptet-luxR-(OR1)
-
⑤Prom-luxR-(OR2)
+
  ⑤Prom-luxR-(OR2)
-
⑥Prom-luxR-(OR1)
+
  ⑥Prom-luxR-(OR1)
-
1. ①1㎕
+
1. ①1uL, ③3uL
-
③3㎕
+
2. ②1uL, ④4uL
-
2. ②1㎕
+
3. ①1uL, ⑤1uL
-
④4㎕
+
  4. ②1uL, ⑥1uL
-
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㎕
+
-
 
+
-
94°C – 5min
+
-
94°C – 15sec
+
-
52°C – 30sec      10cycles
+
-
72°C – 1min
+
-
72°C – 10min
+
-
----------------
+
-
4°C  stop
+
 +
1. ①-③
 +
2. ②-④
 +
  --------------------------
 +
  template    1uL-4uL
 +
  primer Fwd  -
 +
  primer Rev  -
 +
  dNTP        5uL
 +
  10xbuffer  5uL
 +
  NFW        34uL
 +
  VentP      1uL
 +
  -------------------------
 +
              50uL<br>
 +
3. ①-⑤
 +
4. ②-⑥
 +
  -------------------------
 +
  template      1uL-1uL
 +
  primer Fwd    -
 +
  primer Rev    -
 +
  dNTP          5uL
 +
  10xbuffer    5uL
 +
  NFW          37uL
 +
  VentP        1uL
 +
  -------------------------
 +
                50uL<br>
 +
5. control
 +
  -------------------------
 +
  template      1uL
 +
  primer Fwd    5uL
 +
  primer Rev    5uL
 +
  dNTP          5uL
 +
  10xbuffer    5uL
 +
  NFW          28uL
 +
  VentP        1uL
 +
  -------------------------
 +
                50uL<br>
 +
PCR
 +
  94°C – 5min
 +
  94°C – 15sec    --
 +
  52°C – 30sec      │- 10cycles
 +
  72°C – 1min      --
 +
  72°C – 10min
 +
  ----------------
 +
  4°C  stop
<html><h3>2010-09-20</h3></html>
<html><h3>2010-09-20</h3></html>
-
transformation
+
Transformation
-
  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㎕
+
11:30 ~  DNA : 1uL   XL10GkanR : 30uL   SOC : 100uL<br>
-
 
+
  Cm             Amp
-
 
+
-
  Cm             Amp
+
  1-3C            1-1K
  1-3C            1-1K
  1-3A            pSB1A3
  1-3A            pSB1A3
-
  pSB1C3
+
  pSB1C3<br>
-
 
+
liquid incubation(37°C) 7:15~
-
<liquid incubation(37°C) 7:15~>
+
Mini-prep
-
 
+
ZYMO purification
-
 
+
From PCR product from SOEing PCR (09-19)
-
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
-
  ZYMO purification
+
to 2mL tube with column. After operating centrifuge, transfer remaining a little PCR product to 2mL tube with column.
-
      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
-
      2. centrifuge 1min → throwing away left solution
+
3. Same to mini-prep
-
      3. Same to mini-prep
+
   * wash buffer 200uL -> centrifuge 30sec -> throwing away left solution.  X2
-
   * wash buffer 200㎕ -> centrifuge 30sec -> throwing away left solution.  X2
+
   * wash buffer 200uL -> centrifuge 1min -> throwing away left solution.  X1
-
   * wash buffer 200㎕ -> 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>
-
↓
+
PCR
PCR
-
 
+
PCR Condition
-
 
+
  94°C – 5min
-
94°C – 5min
+
  94°C – 15sec     --
-
94°C – 15sec
+
  52°C – 30sec      │-10 cycles
-
52°C – 30sec      10 cycles
+
  72°C – 1min     --
-
72°C – 1min
+
  72°C – 10min
-
72°C – 10min
+
  ----------------
-
----------------
+
  4°C  stop<br>
-
4°C  stop
+
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      10uL
-
template      10㎕
+
  primer Fwd    5uL
-
primer Fwd    5㎕
+
  primer Rev    5uL
-
primer Rev    5㎕
+
  dNTP           5uL
-
dNTP           5㎕
+
  10xbuffer     5uL
-
10xbuffer       5㎕
+
  NFW           19uL
-
NFW         19㎕
+
  VentP         1uL
-
VentP           1㎕
+
  -------------------------
-
-------------------------
+
                50uL<br>
-
              50㎕
+
Using primer
-
 
+
  1. ①-③ Fwd : Ptet-luxR∙FA-R  Rev : TcIg∙FA-R
-
To use primer
+
  2. ②-④ Fwd : Ptet-luxR∙FA-R  Rev : TcIg∙FA-R
-
1. ①-③ Fwd : Ptet-luxR∙FA-R  Rev : TcIg∙FA-R
+
  3. ①-⑤ Fwd : PluxR∙FA-R    Rev : TcIg∙FA-R
-
2. ②-④ Fwd : Ptet-luxR∙FA-R  Rev : TcIg∙FA-R
+
  4. ②-⑥ Fwd : PluxR∙FA-R    Rev : TcIg∙FA-R
-
3. ①-⑤ Fwd : PluxR∙FA-R    Rev : TcIg∙FA-R
+
  5. control<br>
-
4. ②-⑥ Fwd : PluxR∙FA-R    Rev : TcIg∙FA-R
+
Using template
-
5. control
+
  1. Ptet-LuxR-PT7/cI(OR2)-GFP
-
 
+
  2. Ptet-LuxR-PT7/cI(OR1)-GFP
-
To use template
+
  3. Prom-LuxR-Pt7/cI(OR2)-GFP
-
1. Ptet-LuxR-PT7/cI(OR2)-GFP
+
  4. Prom-LuxR-Pt7.cI(OR1)-GFP
-
2. Ptet-LuxR-PT7/cI(OR1)-GFP
+
  5. control<br>
-
3. Prom-LuxR-Pt7/cI(OR2)-GFP
+
PCR condition(usingTAKARA PCR thermal cycler)
-
4. Prom-LuxR-Pt7.cI(OR1)-GFP
+
  94°C – 5min
-
5. control
+
  94°C – 15sec     --
-
 
+
  51°C – 30sec      │-25 cycles
-
PCR condition(usingTAKARA PCR thermal cycler)
+
  72°C – 1min     --
-
94°C – 5min
+
  72°C – 10min
-
94°C – 15sec
+
↓<br>
-
51°C – 30sec      25 cycles
+
Gel Electrophoresis
-
72°C – 1min
+
Cm            Amp
-
72°C – 10min
+
-
 
+
-
↓
+
-
gel electrophoresis
+
-
 
+
-
 
+
-
 
+
-
 
+
-
Cm            Amp
+
  1-3C            1-1K
  1-3C            1-1K
  1-3A            pSB1A3
  1-3A            pSB1A3
  pSB1C3
  pSB1C3
-
 
+
↓<br>
-
<liquid incubation(37°C) 7:15~>
+
Liquid Incubation(37°C) 7:15~
-
↓
+
-------------------------
-
mini-prep
+
template       3uL
-
 
+
primer Fwd    5uL
-
-------------------------
+
primer Rev    5uL
-
template     3㎕
+
dNTP           5uL
-
primer Fwd    5㎕
+
10xbuffer     5uL
-
primer Rev    5㎕
+
NFW           26uL
-
dNTP           5㎕
+
VentP         1uL
-
10xbuffer       5㎕
+
-------------------------
-
NFW         26㎕
+
                50uL<br>
-
VentP           1㎕
+
Using primer
-
-------------------------
+
-
              50㎕
+
-
 
+
-
To use primer
+
   Fwd : pSB1C3FA-F
   Fwd : pSB1C3FA-F
-
   Rev : pSB1C3FA-R
+
   Rev : pSB1C3FA-R<br>
-
 
+
Using template
-
To use template
+
   1. 1-3C(J04450,pSB3C5)
   1. 1-3C(J04450,pSB3C5)
   2. pSB1C3
   2. pSB1C3
   3. 1-3A(J04450,pSB1C3)
   3. 1-3A(J04450,pSB1C3)
   4. 1-1K(J04450,J63010)
   4. 1-1K(J04450,J63010)
-
   5. control(pCI-GFP)  primer Fwd : PcI-GFP∙fwd Rev : PcI-GFP∙rev
+
   5. control(pCI-GFP)  primer Fwd : PcI-GFP∙fwd Rev : PcI-GFP∙rev<br>
-
 
+
PCR condition
-
PCR condition
+
  94°C – 5min
-
94°C – 5min
+
  94°C – 15sec   --
-
94°C – 15sec
+
   51°C – 30sec     │-25 cycles
-
51°C – 30sec      25 cycles
+
  72°C – 1min    --
-
72°C – 1min
+
   72°C – 10min
-
72°C – 10min
+
↓<br>
-
↓
+
Gel Electrophoresis
-
gel electrophoresis
+
-
 
+
-
 
+
-
<html><h3>2010-09-21</h3></html>
+
-
   Because it didn’t come out vector PCR(gel electrophoresis), we operated re-experiment to change template and template amounts.
+
-
 
+
-
To use template
+
-
1. Biobrick 2010 1-3A(pSB1C3)
+
-
2. Biobrick 2010 pSB1C3
+
-
3. Biobrick 2010 2-9A(pSB1A3)
+
-
4. control
+
-
 
+
-
To use primer
+
-
Fwd : pSB1C3 FASTR-F
+
-
Rev : pSB1C3 FASTR-R
+
-
 
+
-
-------------------------
+
-
template      5㎕
+
-
primer Fwd    5㎕
+
-
primer Rev    5㎕
+
-
dNTP            5㎕
+
-
10xbuffer        5㎕
+
-
NFW          24㎕
+
-
VentP          1㎕
+
-
-------------------------
+
-
              50㎕
+
-
 
+
-
PCR(TAKARA)
+
-
94°C – 5min
+
-
94°C – 15sec
+
-
51°C – 30sec       25 cycles
+
-
72°C – 1min
+
-
72°C – 10min
+
-
 
+
-
Vector PCR
+
-
To use template
+
-
1. Biobrick 2010 1-3A(pSB1C3)
+
-
2. Biobrick 2010 pSB1C3
+
-
3. Biobrick 2010 2-9A(pSB1A3)
+
-
4. control
+
-
 
+
-
-------------------------
+
-
template      3㎕
+
-
primer Fwd     5㎕
+
-
primer Rev    5㎕
+
-
dNTP            5㎕
+
-
10xbuffer        5㎕
+
-
NFW          26㎕
+
-
VentP          1㎕
+
-
-------------------------
+
-
              50㎕
+
-
 
+
-
To use primer
+
-
   Fwd : pSB1C3 FASTR-F
+
-
  Rev : pSB1C3 FASTR-R
+
-
 
+
-
 
+
-
PCR
+
-
94°C – 5min
+
-
94°C – 30sec
+
-
51°C – 30sec      25 cycles
+
-
72°C – 2min
+
-
72°C – 10min
+
-
 
+
-
 
+
-
<html><h3>2010-09-22</h3></html>
+
-
  Biobrick 2010 2-9A(pSB1A3) -> gel extraction
+
-
 
+
-
gel electrophoresis - Biobrick 2010 2-9A(pSB1A3), Biobrick 2010 1-3A(pSB1C3),
+
-
Biobrick 2010 pSB1C3, Biobrick 2010 2-9A(pSB1A3)
+
-
  As a result, it didn’t come out band of 1-3A and pSB1C3. We concluded re-experiment from liquid incubation to mini-prep. Must check gel electrophoresis band after mini-prep.
+
-
 
+
-
  FASTR Cloning (Plasmid1)
+
-
  Vector (about 2kbp)
+
-
- pSB1A3 (conservative solution 100ng/㎕)
+
-
  Insert (about 1kbp)
+
-
  1. Pet-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕)
+
-
  2. Pet-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕)
+
-
  3. Prom-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕)
+
-
4. Prom-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕)
+
-
 
+
-
master mix
+
-
---------------------------------------------------------------------
+
-
Vector(50ng)        0.5 2
+
-
Insert(75ng) 3 -
+
-
Tango buffer 0.5 2
+
-
T4 Ligase buffer 0.5 2
+
-
ATP 1 4
+
-
LguI 0.5 2
+
-
Ligase 0.5 2
+
-
DpnI 0.5 2
+
-
NFW 3 12
+
-
-----------------------------------------------------------------------
+
-
Total 10㎕ 28㎕
+
-
Room temperature 2h
+
-
 
+
-
  Transformation
+
-
  To use cell strain : BL21(DE3) 50㎕
+
-
  To use plasmid
+
-
      L1 : Pet-LuxR-PT7/cI(OR2)-GFP  5㎕
+
-
      L2 : Pet-LuxR-PT7/cI(OR1)-GFP  5㎕
+
-
      L3 : Prom-LuxR-PT7/cI(OR2)-GFP 5㎕
+
-
L4 : Prom-LuxR-PT7/cI(OR1)-GFP 5㎕
+
-
 
+
-
  After transformation
+
-
    We added IPTG(0μM / 100μM / 200μM) to each cell strain. It became 12 plates.
+
-
 
+
-
  ※A Reason of using DE3 cell strain
+
-
DE3 cell strain has T7 RNA Polymerase gene in genome, and under IPTG derivation T7RNAP is expressed. Plasmid1 has T7/cI hybrid promoter. So if this promoter is accelerated by T7RNAP, GFP is expressed and shines green. In other words, we used DE3 cell strain to check T7 accelerating function of PT7/cI.
+
-
 
+
-
<html><h3>2010-09-23</h3></html>
+
-
  From yesterday, we did mini-prep liquid incubated sample 1-3A and pSB1C3. And then it is eluted by 50㎕ Elution Solution.
+
-
  ↓
+
-
gel electrophoresis(1㎕)
+
-
 
+
-
 
+
-
L1 : Pet-LuxR-PT7/cI(OR2)-GFP  5㎕
+
-
    L2 : Pet-LuxR-PT7/cI(OR1)-GFP  5㎕
+
-
    L3 : Prom-LuxR-PT7/cI(OR2)-GFP 5㎕
+
-
L4 : Prom-LuxR-PT7/cI(OR1)-GFP 5㎕
+
-
 
+
-
Remaining of above sample is transformed and incubated.
+
-
cell strain : XL10G(50㎕/tube)
+
-
plasmid : L1~L4(total 4)
+
-
 
+
-
FASTR Cloning (Plasmid1)
+
-
  Vector (about 2kbp)
+
-
- pSB1A3 (conservative solution 100ng/㎕)
+
-
  Insert (about 1kbp)
+
-
  1. Pet-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕)
+
-
  2. Pet-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕)
+
-
  3. Prom-LuxR-PT7/cI(OR2)-GFP (conservative solution 25ng/㎕)
+
-
4. Prom-LuxR-PT7/cI(OR1)-GFP (conservative solution 25ng/㎕)
+
-
 
+
-
master mix
+
-
---------------------------------------------------------------------
+
-
Vector(50ng)        0.5 2
+
-
Insert(75ng) 3 -
+
-
Tango buffer 0.5 2
+
-
T4 Ligase buffer 0.5 2
+
-
ATP 1 4
+
-
LguI 0.5 2
+
-
Ligase 0.5 2
+
-
DpnI 0.5 2
+
-
NFW 3 12
+
-
-----------------------------------------------------------------------
+
-
Total 10㎕ 28㎕
+
-
Room temperature 2h
+
-
 
+
-
After ligation plasmid is transformed and incubated in LB broth.
+
-
cell strain : BL21(DE3), XL10G(each 50㎕/tube)
+
-
plasmid : L1~L4
+
-
 
+
-
 
+
-
 
+
-
+
-
<html><h3>2010-09-24</h3></html>
+
-
 
+
-
At 23th September
+
-
Remaining of above sample is transformed and incubated.
+
-
cell strain : XL10G(50㎕/tube)
+
-
plasmid : L1~L4(total 4)
+
-
 
+
-
We picked 4 colonies of each plate out from L1~L4 plates and operated colony PCR. Moreover, we did liquid incubation each colony.
+
-
 
+
-
Insert Check Colony PCR(Plasmid 1)
+
-
master mix
+
-
--------------------------------------------------------------------------
+
-
Template(picked colonies)
+
-
Primer VF 2㎕ 40㎕
+
-
Primer VR 2㎕ 40㎕
+
-
10x Buffer 2㎕ 40㎕
+
-
dNTP 2㎕ 40㎕
+
-
NFW 10㎕ 200㎕
+
-
Taq DNA Polymerase 0.5㎕ 5㎕
+
-
--------------------------------------------------------------------------
+
-
Total 20㎕ 400㎕
+
-
 
+
-
↓
+
-
PCR
+
-
94°C – 5min
+
-
94°C – 30sec
+
-
51°C – 30sec        20 cycles
+
-
72°C – 1min
+
-
72°C – 10min
+
-
↓
+
-
gel electrophoresis
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
-
 
+
==Notebook==
==Notebook==
-
8/31 We've started experiment. First, we researched the property of T7 promoter.<br>
+
8/31 We've started experiment. First, we researched the property of T7 promoter.<br>
-
9/5 We are researching promoter and inverters.<br>
+
9/5 We are researching promoter and inverters.<br>
-
10/7 We are on the bench!! Design primers, PCR and experimentsssssssssss :D
+
10/7 We are on the bench!! Design primers, PCR and experimentsssssssssss :D

Latest revision as of 19:10, 27 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
Retrieved from "http://2010.igem.org/Team:Chiba/Notebook_1"