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PCR Strategy for Splotch Mice
Reagents
1. Primers (100 µM in H20)
SP F intron 3.forward,5'-GAG AGG GTT GAG TAC GTT AGC-3';
SP R+ Wild type,reverse.5'-CGG CTG ATA GAA CTC ACT GGA-3';
SP R_ mutant reverse. 5'-CGG CTG ATA GAA CTC ACA CAC-3';
SP F and SPR+=wild Type
SP F and SPR_=Mutant SP
PCR product. 110bp
2. Example Reaction
10 x PCR Buffer 10λ
25mM dNTP's 0.8λ
primer SP F 0.5λ
SP R+ or SP R- 0.5λ
Taq Polymerase 0.5λ
H2O 87.7λ
Total 100λ
Aliquot 30λ to each tube. Add 1.0λ DNA to the reaction tube.
3. Program
94°C 1 Min
60°C 1 Min
72°C 1 Min
go to step 1 repeat 35 cycles
72°C 5 Min
4°C 99hrs
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