Tempel Lab Protocols

BAX GENOTYPE

You will Need

Mouse Tail Digests

3 Primers:
661 Common Forward
662 Neo Reverse
663 WT Reverse

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

661 GTT GAC CAG AGT GGC GTA GG
662 CCG CTT CCA TTG CTC AGC GG
663 GAG CTG ATC AGA ACC ATC ATG

Procedure
Run the wildtype (661 and 663 primers) and the mutant (661 and 662 primers) PCR reactions in seperate tubes.

PCR Rxn
Sterile Water 17.8 ul
10x PCR buffer w/MgCl2 2.5ul
10mM Nucleotide mix 0.5ul
BAX Com F primer (20 uM) 1 ul
BAX Reverse Primer

Neo R (20 uM) WT R (20 uM)

 1 ul
FastStart Taq 0.2ul
Tail Lysate 2 ul
Total Volume 25ul
PCR Protocol

94 deg C 4:30
94 deg C
 0:30
63 deg C
 0:45 x35
72 deg C
 0:45
72 deg C 7:00
4 deg C Hold

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

Bands
WildType: 304 bp
Mutant: 507 bp