The
Jacks Lab
protocols
Kras G12D Conditional
PCR
(multiplex & single allele)
NOTE: this reaction looks for the presence of the stop cassette NOT the mutation.
Samples must be sequenced to look for mutation.
The guarantee for this PCR to work is to use
"Red Taq Ready Mix" from Sigma, cat. no. R2523.
Alternatively, use:
Reaction Mix:
| Tail DNA | 1.0ul |
| 10x PCR buffer | 2.0ul |
| dt5-(40pmol/ul)* | 0.6ul |
| Uni'-(40pmol/ul) * | 0.4ul |
| SD5'-(40pmol/ul)* | 0.2ul |
| milliQ | 14.8ul |
| Taq (1/25) | 1.0ul |
*or for single allele reactions use 10pmol primers per reaction
10X PCR Buffer:
| 500 mM KCl |
| 100mM Tris pH 8.3 |
| 15 mM MgCl2 |
| 1 mg/ml BSA |
| 2 mM dNTPs |
| dH20 |
(store at -20† C)
PCR Primers:
| dt5' new (5'wt) | 5' GTC GAC AAG CTC ATG CGG G 3' |
| Uni'new (3'universal) | 5' CGC AGA CTG TAG AGC AGC G 3’ |
| SD5'-new (5'mut) | 5’ CCA TGG CTT GAG TAA GTC TGC 3’ |
PCR Conditions:
| 1. | 94C | 3:00 |
| 2. | 94C | 0:30 |
| 3. | 60C | 1:30 |
| 4. | 72C | 1:00 |
| 5. | Goto 2 x34 cycles | |
| 6. | 72C | 5:00 |
| 7. | 4C | 0:00 |
| END | ||
Expected Bands:
| Wt= ~500 bp |
| Mut= ~550 bp |