The Jacks Lab
protocols
p53 unrecombined point-mutant alleles PCR
Reaction Mix:
| Tail DNA | 1.0 無 |
| 10x buffer | 2.0ul |
| SD5' (20uM) | 0.2無 |
| DT050500.2 (20uM) | 0.5無 |
| DT011200.3 (20uM) | 0.5無 |
| dH20 | 14.8 無 |
| Taq (1/25) | 1.0ul |
PCR Primers:
| SD5' | 5' AGC TAG CCA CCA TGG CTT GAG TAA GTC TGC A3' |
| DT050500.2 | 5' TTA CAC ATC CAG CCT CTG TGG 3' |
| DT011200.3 | 5' CTT GGA GAC ATA GCC ACA CTG 3' |
PCR Conditions:
| 1. | 94C | 3:00 |
| 2. | 94C | 1:00 |
| 3. | 60C | 1:30 |
| 4. | 72C | 2:00 |
| 5. | Goto 2 x30 cycles | |
| 6. | 72C | 5:00 |
| 7. | 4C | 0:00 |
| 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:
| Control (for PCR) = ~170 bp Note: Does not distinguish between heterozygotes and homozygotes! Only screens for LSL cassette! To detect point mutation, have to sequence! |
| LSL= ~270 bp |