Team:Freiburg/Notebook/TAL

From 2012.igem.org

(Difference between revisions)
(Our way to the TAL vector)
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<table border="0" style="background-color:transparent">
<table border="0" style="background-color:transparent">
<tr>
<tr>
-
<th></th>
+
<th></th><th>Tube 1 [µl]</th><th>Tube 2 [µl]</th>
-
<th>Tube 1 [µl]</th>
+
</tr><tr>
-
<th>Tube 2 [µl]</th>
+
<td>Primer MutPCR fo [10 mM]</td><td>1</td><td>0,25</td>
-
</tr>
+
</tr><tr>
-
<tr>
+
<td>Primer MutPCR re [10 mM]</td><td>1</td><td>0,25</td>
-
<td>Primer MutPCR fo [10 mM]</td>
+
</tr><tr>
-
<td>1</td><td>0,25</td>
+
<td>dNTPs [10 mM]</td><td>1</td><td>1</td>
-
</tr>
+
</tr> <tr>
-
<tr>
+
<td>Phusion Buffer HF</td><td>1,25</td><td>1,25</td>
-
<td>Primer MutPCR re [10 mM]</td>
+
</tr><tr>
-
<td>1</td><td>0,25</td>
+
<td>Phusion Polymerase</td><td>0,25</td><td>0,25</td>
-
</tr>
+
</tr> <tr>
-
<tr>
+
<td>DMSO</td><td>1</td><td>1</td>
-
<td>dNTPs [10 mM]</td>
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</tr> <tr>
-
<td>1</td><td>1</td>
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<td>MgCl2</td><td>1</td><td>1</td>
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</tr>
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</tr> <tr>
-
<tr>
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<td>Template</td><td>1</td><td>1</td>
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<td>Phusion Buffer HF</td>
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</tr> <tr>
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<td>1,25</td><td>1,25</td>
+
<td>ddH2O</td><td>5</td><td>6,5</td>
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</tr>
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</tr></table>
-
<tr>
+
-
<td>Phusion Polymerase</td>
+
-
<td>0,25</td><td>0,25</td>
+
-
</tr>
+
-
<tr>
+
-
<td>DMSO</td>
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-
<td>1</td><td>1</td>
+
-
</tr>
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-
<tr>
+
-
<td>MgCl2</td>
+
-
<td>1</td><td>1</td>
+
-
</tr>
+
-
<tr>
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-
<td>Template</td>
+
-
<td>1</td><td>1</td>
+
-
</tr>
+
-
<tr>
+
-
<td>ddH2O</td>
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-
<td>5</td><td>6,5</td>
+
-
</tr>
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-
</table>
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<br>
<br>
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<table border="0" width="20%" style="background-color:transparent">
<table border="0" width="20%" style="background-color:transparent">
<tr>
<tr>
-
<td>1.</td>
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<td>1.</td><td>95°C</td><td>5min</td>
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<td>95°C</td><td>5min</td>
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</tr><tr>
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</tr>
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<td>2.</td><td>95°C</td><td>50s</td>
 +
</tr><tr>
 +
<td>3.</td><td>80°C</td><td>50s</td>
 +
</tr><tr>
 +
<td>4.</td><td>72°C</td><td>3min</td>
 +
</tr><tr>
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<td>5.</td><td>72°C</td><td>7min</td>
 +
</tr><tr>
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<td>6.</td><td>4°C</td><td>//</td>
 +
</tr></table><br><br>
 +
 
 +
2.) Transformation of Sirt2 and HAT5 in DH10B starin of E. coli using our standard protocol for transformation. Kanamycin resistance was used<br><br>
 +
 
 +
3.) GGC in order to produce our Mammobrick:<br><br>
 +
 
 +
Program for Thermocycler (20 cycles)
 +
 
 +
<table border="0" width="40%" style="background-color:transparent">
<tr>
<tr>
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<td>2.</td>
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<th>Contens</th><th>[µl]</th>
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<td>95°C</td><td>50s</td>
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</tr><tr>
-
</tr>
+
<td>BsaI [10 U/µl]</td><td>1,5</td>
-
<tr>
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</tr><tr>
-
<td>3.</td>
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<td>NEB Buffer 4</td><td>95°C</td><td>50s</td>
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<td>80°C</td><td>50s</td>
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</tr><tr>
-
</tr>
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<td>ATP [10 mM]</td><td>1</td>
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<tr>
+
</tr><tr>
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<td>4.</td>
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<td>BSA</td><td>1</td>
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<td>72°C</td><td>3min</td>
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</tr><tr>
-
</tr>
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<td>TALEN fragment [40 ng]</td><td>2</td>
-
<tr>
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</tr><tr>
-
<td>5.</td>
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<td>exCMV promotor [20 ng]</td><td>0,5</td>
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<td>72°C</td><td>7min</td>
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</tr><tr>
-
</tr>
+
<td>PostORF [20 ng]</td><td>1,25</td>
 +
</tr><tr>
 +
<td>PuroORF [20 ng]</td><td>0,5</td>
 +
</tr><tr>
 +
<td>T7 Ligase</td><td>0,25</td>
 +
</tr><tr>
 +
<td>DTT [10 mM]</td><td>1</td>
 +
</tr><tr>
 +
<td>ddH2O</td><td>1</td>
 +
</tr></table><br><br>
 +
 
 +
<table border="0" width="40%" style="background-color:transparent">
<tr>
<tr>
-
<td>6.</td>
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<th>Program for Thermocycler (15 cycles)</th>
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<td>4°C</td><td>//</td>
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</tr><tr>
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</tr>
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<td>1. &nbsp;&nbsp;&nbsp;&nbsp; 37°C &nbsp;&nbsp;&nbsp;&nbsp; 5min</td>
-
</table>
+
</tr><tr>
 +
<td>2. &nbsp;&nbsp;&nbsp;&nbsp; 20°C &nbsp;&nbsp;&nbsp;&nbsp; 5min</td>
 +
</tr></table>

Revision as of 16:38, 26 September 2012





Labbook - TAL vector



Notebooksymbol.png


08/08/12

1.) MutPCR for pSB1C3 vector backbone in order to remove restriction sites vor BsmBI
2.) Transformation of Sirt2 and HAT5
3.) GGC in order to produce our MammoBrick

1.) MutPCR:

Tube 1 [µl]Tube 2 [µl]
Primer MutPCR fo [10 mM]10,25
Primer MutPCR re [10 mM]10,25
dNTPs [10 mM]11
Phusion Buffer HF1,251,25
Phusion Polymerase0,250,25
DMSO11
MgCl211
Template11
ddH2O56,5


Program for Thermocycler (20 cycles)

1.95°C5min
2.95°C50s
3.80°C50s
4.72°C3min
5.72°C7min
6.4°C//


2.) Transformation of Sirt2 and HAT5 in DH10B starin of E. coli using our standard protocol for transformation. Kanamycin resistance was used

3.) GGC in order to produce our Mammobrick:

Program for Thermocycler (20 cycles)

Contens[µl]
BsaI [10 U/µl]1,5
NEB Buffer 495°C50s
ATP [10 mM]1
BSA1
TALEN fragment [40 ng]2
exCMV promotor [20 ng]0,5
PostORF [20 ng]1,25
PuroORF [20 ng]0,5
T7 Ligase0,25
DTT [10 mM]1
ddH2O1


Program for Thermocycler (15 cycles)
1.      37°C      5min
2.      20°C      5min