p53 +/- PCR

Reaction Mix:

Tail DNA 1.0 µL
10x buffer (with NTPs) 2.0ul
p53X6.5 (20uM) 0.4 µL
p53X7 (20uM) 0.4 µL
Neo18.5 (20uM) 0.4 µL
dH20 14.8 µL
Taq (1/25) 1.0ul

PCR Primers:

p53X6.5 5' ACA gCg Tgg Tgg TAC CTT AT 3'
p53X7 5' TAT ACT CAg AgC Cgg CCT 3'
Neo18.5 5' CTA TCA ggC ATA gCg TTg g 3'

PCR Conditions:

1. 94C 3:00
2. 94C 1:00
3. 60C 2:00
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:

Wild type (gene intact) = 430 bp on 1-2% gel
Mutant (gene knockout) = 590 bp