Kras KO PCR

Reaction Mix:

Tail DNA 4.0ul
10x PCR buffer 2.0ul
k-5'-IO (20uM) 0.2ul
K-3'-1a (20uM) 0.1ul
K-3'-neo (20uM) 0.25ul
milliQ 12.45ul
Taq (1/25) 1.0ul

PCR Primers:

k-5'-IO (5'WT) 5' AGG GTA GGT GTT GGG ATA GC 3'
K-3'-1a (3'WT) 5’ CTC AGT CAT TTT CAG CAG GC 3’
K-3'-neo (3'MUT) 5’ ACG AGA CTA GTG AGA CTG GC 3’

PCR Conditions:

1. 94C 3:00
2. 94C 1:00
3. 60C 1:00
4. 72C 1:00
5. Goto 2 x33 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:

Wt= ~360 bp
Mut= ~270 bp