Preparation of PCR Material

1. 10X Taq buffer: 100 mM Tris-HCl, 15 mM MgCl2, 500 mM KCl, pH 8.3.

2. 25 mM dNTP mix: 1:1:1:1 mix of 100 mM dATP, dCTP, dGTP, and dTTP, (Boehringer Mannheim, Mannheim, Germany), store in 50-|L aliquots at -20°C.

3. Oligonucleotides (Pharmacia, Piscataway, NJ):

NoildT: cat ctc gag cgg cgg ctt ttt ttt ttt ttt ttt ttt ttt t: Primary PCR only. NoiI24: gcg gcc gct ttt ttt ttt ttt ttt: Amplification of driver. NoilUnique: cat ctc gag cgg ccg ctt ttt ttt : Alternative tracer recovery oligo. T7dT: gat ctg cac gcg taa tac gac tcg agt ttt ttt ttt ttt ttt: Amplification of tracer. T7Unique: gat ctg cac gcg taa tac gac tc: Recovery of tracer after subtraction.

5. Taq polymerase (Boehringer Mannheim).

6. PCR reaction mix: 1X Taq polymerase reaction buffer, 0.25 mM dNTP, 0.5 OD260/mL oligonucleotide, 1.2 U/100 |L Taq polymerase.

7. 1.7% (w/v) agarose gel (SeaKem, FMC BioProducts, Rockland, ME).

9. Ethidium bromide (10 mg/mL) (as a possible carcinogen, adequate safety precautions must be taken).

10. PCR product of known concentration.

11. Qiagen Tip-20.

14. QF buffer: 1.2 M NaCl, 50 mM MOPS, pH 8.0, 15% ethanol.

15. QIAspin PCR purification kit (Qiagen, Chatsworth, CA).

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