Koeris/Notebook/2007-1-19: Difference between revisions
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| Line 29: | Line 29: | ||
#4 deg C - indefinite | #4 deg C - indefinite | ||
==Gel image== | ==Gel image== | ||
Loaded | Loaded 10ul, i.e. 10% of Rx on the gel; 10ul 2-log ladder as well | ||
==Restriction digest== | ==Restriction digest== | ||
*Use Qiagen PCR cleanup kit to remove salts and enzymes | *Use Qiagen PCR cleanup kit to remove salts and enzymes | ||
*Digest PCR amplicons with KpnI, Hind III - double digest | *Digest PCR amplicons with KpnI, Hind III - double digest | ||
Revision as of 12:00, 19 January 2007
Re-cloning of [His]6
Due to lack of material, I could not recover the A plasmid and have to re-clone it.
PCR set-up
- 100 ul total reaction volume
- 1ul template DNA @ ~40ng/ul
- 200pmol primer mix
- 97ul Taq SuperMix
Pipetting scheme
Labels in the respective field codes
| Primer | Tube Label |
|---|---|
| A his6 2F&R 200pmol | A1 |
| A his6 2F&R 200pmol | A2 |
Thermal profile
- 92 deg C - 5min
- 92 deg C - 30s
- 50 deg C - 45s
- 72 deg C - 120s
- Cycle back to 2. 29X
- 72 deg C - 10min
- 4 deg C - indefinite
Gel image
Loaded 10ul, i.e. 10% of Rx on the gel; 10ul 2-log ladder as well
Restriction digest
- Use Qiagen PCR cleanup kit to remove salts and enzymes
- Digest PCR amplicons with KpnI, Hind III - double digest
