"
Team:Freiburg/Notebook/TAL
From 2012.igem.org
(Difference between revisions)
Pablinitus (Talk | contribs) (→Our way to the TAL vector) |
|||
| Line 17: | Line 17: | ||
<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> | + | </tr> <tr> |
| - | <td>1</td><td>1</td> | + | <td>MgCl2</td><td>1</td><td>1</td> |
| - | </tr> | + | </tr> <tr> |
| - | <tr> | + | <td>Template</td><td>1</td><td>1</td> |
| - | <td>Phusion Buffer HF</td> | + | </tr> <tr> |
| - | <td>1,25</td><td>1,25</td> | + | <td>ddH2O</td><td>5</td><td>6,5</td> |
| - | </tr> | + | </tr></table> |
| - | <tr> | + | |
| - | <td>Phusion Polymerase</td> | + | |
| - | <td>0,25</td><td>0,25</td> | + | |
| - | </tr> | + | |
| - | <tr> | + | |
| - | <td>DMSO</td> | + | |
| - | <td>1</td><td>1</td> | + | |
| - | </tr> | + | |
| - | <tr> | + | |
| - | <td>MgCl2</td> | + | |
| - | <td>1</td><td>1</td> | + | |
| - | </tr> | + | |
| - | <tr> | + | |
| - | <td>Template</td> | + | |
| - | <td>1</td><td>1</td> | + | |
| - | </tr> | + | |
| - | <tr> | + | |
| - | <td>ddH2O</td> | + | |
| - | <td>5</td><td>6,5</td> | + | |
| - | </tr> | + | |
| - | </table> | + | |
<br> | <br> | ||
| Line 64: | Line 43: | ||
<table border="0" width="20%" style="background-color:transparent"> | <table border="0" width="20%" style="background-color:transparent"> | ||
<tr> | <tr> | ||
| - | <td>1.</td> | + | <td>1.</td><td>95°C</td><td>5min</td> |
| - | <td>95°C</td><td>5min</td> | + | </tr><tr> |
| - | </tr> | + | <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> | ||
| + | <td>5.</td><td>72°C</td><td>7min</td> | ||
| + | </tr><tr> | ||
| + | <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> | ||
| - | <td> | + | <th>Contens</th><th>[µl]</th> |
| - | <td>95°C</td><td>50s</td> | + | </tr><tr> |
| - | </tr> | + | <td>BsaI [10 U/µl]</td><td>1,5</td> |
| - | <tr> | + | </tr><tr> |
| - | <td> | + | <td>NEB Buffer 4</td><td>95°C</td><td>50s</td> |
| - | <td> | + | </tr><tr> |
| - | </tr> | + | <td>ATP [10 mM]</td><td>1</td> |
| - | <tr> | + | </tr><tr> |
| - | <td> | + | <td>BSA</td><td>1</td> |
| - | <td> | + | </tr><tr> |
| - | </tr> | + | <td>TALEN fragment [40 ng]</td><td>2</td> |
| - | <tr> | + | </tr><tr> |
| - | <td> | + | <td>exCMV promotor [20 ng]</td><td>0,5</td> |
| - | <td> | + | </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> | ||
| - | < | + | <th>Program for Thermocycler (15 cycles)</th> |
| - | <td> | + | </tr><tr> |
| - | </tr> | + | <td>1. 37°C 5min</td> |
| - | </table> | + | </tr><tr> |
| + | <td>2. 20°C 5min</td> | ||
| + | </tr></table> | ||
Revision as of 16:38, 26 September 2012
Labbook - TAL vector
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] | 1 | 0,25 |
| Primer MutPCR re [10 mM] | 1 | 0,25 |
| dNTPs [10 mM] | 1 | 1 |
| Phusion Buffer HF | 1,25 | 1,25 |
| Phusion Polymerase | 0,25 | 0,25 |
| DMSO | 1 | 1 |
| MgCl2 | 1 | 1 |
| Template | 1 | 1 |
| ddH2O | 5 | 6,5 |
Program for Thermocycler (20 cycles)
| 1. | 95°C | 5min |
| 2. | 95°C | 50s |
| 3. | 80°C | 50s |
| 4. | 72°C | 3min |
| 5. | 72°C | 7min |
| 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 4 | 95°C | 50s |
| ATP [10 mM] | 1 | |
| BSA | 1 | |
| TALEN fragment [40 ng] | 2 | |
| exCMV promotor [20 ng] | 0,5 | |
| PostORF [20 ng] | 1,25 | |
| PuroORF [20 ng] | 0,5 | |
| T7 Ligase | 0,25 | |
| DTT [10 mM] | 1 | |
| ddH2O | 1 |
| Program for Thermocycler (15 cycles) |
|---|
| 1. 37°C 5min |
| 2. 20°C 5min |
