The Jacks Lab

protocols

Nf1 PCR

Reaction Mix:

Tail DNA	2.0ul
10x buffer	2.0ul
NII (20uM)	0.2ul
NIII (20uM)	0.2ul
3'5' (20uM)	0.4ul
milliQ	14.2ul
Taq (1/25)	1.0ul

PCR Primers:

NII	5' GGT ATT GAA TTG AAG CAC 3'
NIII	5’ ATT CGC CAA TGA CAA GAC 3’
3'5'	5’ TTC AAT ACC TGC CCA AGG 3’

PCR Conditions:

1.	94C	3:00
2.	94C	1:00
3.	52C	1:00
4.	72C	2:00
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= ~230 bp

Mut= ~350 bp