From 2010.igem.org
(Difference between revisions)
|
|
| Line 71: |
Line 71: |
| | <STAT1> | | <STAT1> |
| | | | |
| - | <span style=font-size:15px><font face="Maiandra GD"><b>V_STAT1 (expression)</b></font></span><br>
| + | === V_STAT1 (expression) === |
| | | | |
| | <center> | | <center> |
| Line 83: |
Line 83: |
| | </tr> | | </tr> |
| | <tr align=center> | | <tr align=center> |
| - | <td>V_STAT_F</td><td>Forward(NdeI)</td><td>5’-TGTTCATATGGCTAATGTCTCAGTGGTACGAACTTCAG-3’</td><td>24bp</td> | + | <td>V_STAT_F</td><td>Forward(NdeI)</td><td>5’-<font color=blue>TGTT</font><font color=red>CATATG</font><font color=blue>GCTA</font>ATGTCTCAGTGGTACGAACTTCAG-3’</td><td>24bp</td> |
| | </tr> | | </tr> |
| | <tr align=center> | | <tr align=center> |
| - | <td>V_STAT_R</td><td>Reverse(BamHI)</td><td>5’-TATCGGATCCGAATTTACACTTCAGACACAGAAATCAAC-3’</td><td>25bp</td> | + | <td>V_STAT_R</td><td>Reverse(BamHI)</td><td>5’-<font color=blue>TATC</font><font color=red>GGATCC</font><font color=blue>GAAT</font>TTACACTTCAGACACAGAAATCAAC-3’</td><td>25bp</td> |
| | </tr> | | </tr> |
| | </table> | | </table> |
Revision as of 13:37, 10 September 2010
September 1
pREP41 vector has arrived.
-> we will do E.coli transformation and increase its number.
pREP42-GFP vector has arrived (in E.coli)
-> will incubate a day and do spreading for further experiment.
Making culture media
- 500mL LB broth + agar 1.5% (7.5g)
- Do auto clave
- Cool it down until it reaches 50~60℃
- Add ampicillin(1000x 100mg/mL) and stir
- putting out bubbles on culture media
- pouling on petri dish and wait for 1 hour
Culture
- Competent cell + DN10A and leave it in ice for 30min
- Do heat shock for 45 sec and store it in ice for 2min
- Stabilize it in incubator for 30 to 40min
- Do spreading
September ?
<STAT1>
V_STAT1 (expression)
| Primer | Direction | Sequences | Length |
| V_STAT_F | Forward(NdeI) | 5’-TGTTCATATGGCTAATGTCTCAGTGGTACGAACTTCAG-3’ | 24bp |
| V_STAT_R | Reverse(BamHI) | 5’-TATCGGATCCGAATTTACACTTCAGACACAGAAATCAAC-3’ | 25bp |
| Materials | Volume |
| STAT1(KRIBB) (template) | |
| V_STAT_F | |
| V_STAT_R | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| Total | 50ul |
STAT1 (submission)
| Primer | Direction | Sequences | Length |
| STAT_F | Forward | 5’-ATGTCTCAGTGGTACGAACTTCAG-3’ | 24bp |
| STAT_R | Reverse | 5’-TTACACTTCAGACACAGAAATCAAC-3’ | 25bp |
| Materials | Volume |
| STAT1(KRIBB) (template) | |
| STAT_F | |
| STAT_R | |
| dNTP (dNTP mixture 2.5mM TaKaRa) | |
| Taq (Pfu Turbo® DNA Polymerase 2.5U/ μl STRATAGENE) | |
| 10x buffer (10x cloned Pfu Reaction buffer) | |
| DW (3rd sterile water) | |
| Total | 50ul |
September next
|
"
