The Jacks Lab
protocols
P53 conditional PCR (BERNS ALLELE)
P53FLOX
Reaction Mix:
| Tail DNA | 2.0ul |
10x buffer/NTPs |
2.0ul |
| primer A (or C) (10uM) | 0.4 ul |
| primer B (or D) (10uM) | 0.4 ul |
| milliQ | 14.2ul |
| House Taq | 1.0ul |
PCR Primers: Use either primer pair A & B or use primer pair C & D
| A: | 5' CAC AAA AAC AGG TTA AAC CCA G 3' |
| B: | 5' AGC ACA TAG GAG GCA GAG AC 3' |
| C: | 5' AAG GGG TAT GAG GGA CAA GG 3' |
| D: | 5' GAA GAC AGA AAA GGG GAG GG 3' |
PCR Conditions:
| 1. | 94C | 2:00 |
| 2. | 94C | 0:30 |
| 3. | 58C | 0:30 |
| 4. | 72C | 0:50 |
| 5. | Goto 2 x29 cycles | |
| 6. | 72C | 5:00 |
| END | ||
10X PCR Buffer:
| 500 mM KCl |
| 100mM Tris pH 8.3 |
| 15 mM MgCl2 |
| 1 mg/ml BSA |
| 2 mM dNTPs |
| dH20 |
(store at -20C)
Expected Bands (primer pair C & D):
WILD TYPE 431 bp |
LOXP 584 bp |
Expected Bands (primer pair A & B):
| WILD TYPE 288 bp |
LOXP 370 bp |