Reaction Mix:
Tail DNA | 1.0 µL |
10x buffer | 2.0ul |
SD5' (20uM) | 0.2µL |
DT050500.2 (20uM) | 0.5µL |
DT011200.3 (20uM) | 0.5µL |
dH20 | 14.8 µL |
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 |