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Team:ESBS-Strasbourg/Ligation
From 2008.igem.org
(Difference between revisions)
(New page: 1)Remarks: <br> 2)Protocol: <br> -For insert/vector ligation: <br> [http://openwetware.org/wiki/DNA_Ligation see iGEM protocol] -For intra vector ligation: <br> 5 T4 DNA ligase unit,...) |
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2)Protocol: <br> | 2)Protocol: <br> | ||
| - | + | *For insert/vector ligation: <br> | |
[http://openwetware.org/wiki/DNA_Ligation see iGEM protocol] | [http://openwetware.org/wiki/DNA_Ligation see iGEM protocol] | ||
| - | + | *For intra vector ligation: <br> | |
5 T4 DNA ligase unit, for 25-50ng of DNA | 5 T4 DNA ligase unit, for 25-50ng of DNA | ||
| - | + | * ''2c) Protocol according to Fermantas'' | |
| + | ** 50-400 ng vector | ||
| + | ** 1 to 3 times the molar amount of the insert | ||
| + | ** 2 µl ligation buffer | ||
| + | ** 1 µl ligase | ||
| + | ** to 20 µl water | ||
| + | -> vortex and spin down <br> | ||
| + | -> 1h at 22°C water bath (in 4°C room)<br> | ||
| + | -> enzyme inactivation at 65°C for 10 min<br> | ||
| + | -> directly usable for the transformation | ||
[[Team:ESBS-Strasbourg/Protocols|Protocols]] | [[Team:ESBS-Strasbourg/Protocols|Protocols]] | ||
Latest revision as of 08:02, 5 August 2008
1)Remarks:
2)Protocol:
- For insert/vector ligation:
[http://openwetware.org/wiki/DNA_Ligation see iGEM protocol]
- For intra vector ligation:
5 T4 DNA ligase unit, for 25-50ng of DNA
- 2c) Protocol according to Fermantas
- 50-400 ng vector
- 1 to 3 times the molar amount of the insert
- 2 µl ligation buffer
- 1 µl ligase
- to 20 µl water
-> vortex and spin down
-> 1h at 22°C water bath (in 4°C room)
-> enzyme inactivation at 65°C for 10 min
-> directly usable for the transformation
