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CHAPTER 5
MATERIALS AND METHODS
5.2.1.1 TE: tris-Cl (pH 8) + EDTA (ethylene diamine tetra acetate) (Sigma c.
no.E5134)
5.2.1.2 RBC lysis buffer:
0.32M sucrose (to maintain isotonic environment). This solution
should filter prior to use.
10mM tris HCl(pH7.6) (maintain Ph)
5mM MgCl2 (maintain nuclear membrane integrity)
1% Triton 100 (Sigma c.no. t8787)
Make up to 1 L. do not autoclave
5.3 Methodology
5.3.1 Principle
Remove supernant.
Remove supernatant.
Vortex it
Take 5 μL of DNA.
To the master mixture add 1.0 μL of each Primer F (8 pMol) and Primer
R (8 pMol).
Mix well.
Denaturation 94 oC 60sec
32 cycle
Annealing 57 oC 60sec
Elongation 72 oC 60sec
For exon 7, the PCR amplified DNA product is digested with DraI restriction
enzyme and for exon 8, the PCR amplified DNA product is digested with DedI
restriction enzyme.
To the 10 μL of the PCR product add 1 μL of Dra I (10 U/ μL) and 0.5 μL of
the Dde I (5 U/ μL) and keep it for digestion at 37˚C for 24 h. (Procedure
optimized at Institute of Human Genetics, satellite, Ahmedabad)
5.4 Calculation
O.D. at 260nm =
O.D. at 280nm =