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