LONG PCR AMPLIFICATION OF THE FVIII GENE INTRON 22 GENE INVERSION
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AGAROSE
Use a molecular biology grade agarose, one with good gelling strength at low percentages.
DMSO
Use cell culture grade 100% DMSO
Sterile Water
METHOD FOR INTRON 22 LONG RANGE PCR
Samples
1. Extract DNA, determine concentration and use 0.1 to 0.25 µg per reaction. Too much DNA abolishes amplification. It may be advisable to carry out a DNA titration of a specific sample to ensure efficient amplification. If DNA is relatively concentrated then make a 1 in 10 dilution and use between two and five microlitres.
The quality of the DNA is paramount! Use an extraction method that does not shear the DNA and ensure that all traces of phenol are expelled if using phenol extraction. DNA prepared by ammonium acetate salting out methods works very well.
2. Always include a "no DNA" control in PCR
3. Always include a control heterozygous female carrier of the inversion.
Reaction Mix
Each reaction (final volume 25 µl) will require (In reality these will be made as a multiple master mix):
MASTER MIX 1 per reaction: MASTER MIX 2 per reaction:
2.5 µl 10x buffer 2 1.25 µl 10 mM dATP
0.94 µl Taq mix 1.25 µl 10 mM dTTP
