Tempel Lab Protocols

BCL2 GENOTYPE

You will Need

Mouse Tail Digests

2 Primers:
BOR1
BOR2

PCR Reagents:
Sterile water
10X buffer w/MgCl2
10mM Nucleotide mix
FastStart Taq
Primer Sequences

BOR1 CCT GAA ATG AGC CTT GGG ACT GTG AA
BOR2 GTC TCT GAA GAC TCT GCT CAG TTT GG

Procedure

The primers recognize the BCL2 mutant and not the Wildtype. Use a Touchdown PCR program that decreases the primer annealing temperature by 0.3 deg. C per cycle for 25 cycles and then an additional 15 cycles at 58 deg. C.

PCR Rxn
Sterile Water 17.8 ul
10x PCR buffer w/ MgCl2 2.5 ul
10mM Nucleotide mix 0.5 ul
BOR1 primer 20 uM 1 ul
BOR2 primer 20 uM 1 ul
FastStart Taq 0.2 ul
Tail Lysate 2 ul
Total Volume 25 ul
PCR Protocol

94 deg C 4:30
94 deg C
 0:30
66 deg C
0:30
x25
drop by 0.3 degree C per cycle
72 deg C
1:00
94 deg C 0:30
58 deg C 0:30

x15
72 deg C 1:00
72 deg C 7:00
4 deg C Hold

Gel
1.5 % Agarose + 3 ul EtBr (10mg/ml)

Bands
Wildtype: No Band
Mutant: 400-500 bp