Koeris/Notebook/2007-1-19

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

1. 92 deg C - 5min
2. 92 deg C - 30s
3. 50 deg C - 45s
4. 72 deg C - 120s
5. Cycle back to 2. 29X
6. 72 deg C - 10min
7. 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 for 2h