The Jacks Lab

protocols

Kras-LA PCR

Reaction Mix:

Tail DNA	2.0ul
10x PCR buffer	2.0ul
5' Lat WT (20uM)	0.6ul
3' Lat WT (20uM)	0.4ul
3" Lat MUT (20uM)	0.2ul
milliQ	13.8ul
Taq (1/25)	1.0ul

PCR Primers:

5' Lat WT	5' TGC ACA GCT TAG TGA GAC CC 3'
3' Lat WT	5’ GAC TGC TCT CTT TCA CCT CC 3’
3" Lat MUT	5’ GGA GCA AAG CTG CTA TTG GC 3’

PCR Conditions:

1.	94C	3:00
2.	94C	1:00
3.	60C	1:00
4.	72C	0:45
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= ~220 bp

Mut= ~390 bp