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User:DrJones1935/16 July 2012
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I. Ordered Cross-over PCR Primers from Keck
- Primers were ordered with the name/sequence in the following table:
| Name | Tm (Tm overhang/Tm anneal) | Length (bp) | Sequence (overCUTSITEanneal) |
|---|---|---|---|
| cT5_pAf | 66.64 (25.23/58.79) | 30 (10/20) | 5'-tgacTCTAGAtttgctttgtgagcggataa-3' (XbaI) |
| cT5_pAr | 69.08 (30.28/55.61) | 25 (10/15) | 5'-gtaaCTGCAGgagagcgttcaccga-3' (PstI) |
II. Resuspend Lyophilized Primers for Cross-over PCR
- Spin down tubes
- Add the volume specified in the table of TE Buffer to the primer tubes
| Name | Volume (uL) |
|---|---|
| p1f | 208.35 |
| p1r | 217.78 |
| p2f | 199.75 |
| p2r | 199.6 |
| p3f | 199.75 |
| p3r | 190.55 |
| p4f | 248.1 |
| p4r | 149.85 |
| p5f | 127.75 |
| p5r | 199.65 |
- Vortex all tubes briefly
- Spin all tubes down
- Incubate on bench for 10 minutes
- Store at -20 oC
