RAPD Reaction and Cycling Conditions

This protocol was originally derived from the Perkin-Elmer protocol for PCR amplification of DNA and then modified at the Institute of Forest Genetics by various scientists.
March 14, 1994

  Perkin-Elmer reaction:  100 ul reaction scaled down to 25 ul;    10x PCR buffer                                                  25 ul          1x dNTP mix (1.25 mM each dNTP)                     4 ul          200 uM primer [10 uM]                                                     1 ul          0.4 uM Taq polymerase (reduced amt)                   0.2 ul          1.0 units Tween 20                                                       0.13 ul          0.5 %    Optimized for DF:  MgCl (25mM stock)                              3.5 ul          3.5 mM DNA template [8 ng/ul]                           1 ul          8.0 ng H20                                                             x ul Total volume                                          25 ul    Optimized for Taxus:  MgCl (25 mM stock)                             5.5 ul          5.5 mM primer [10uM]                                        0.5 ul          0.2 uM DNA template [8 ng/ul]                            1 ul          8.0 ng    Optimized for P. Brutia:  MgCl (25 mM stock)                             2.2 ul          2.2 mM primer [10uM]                                           1 ul          0.4 uM DNA template [10 ng/ul]                         1 ul         10.0 ng            

Cycling conditions:

  94o for 1 min 37o for 1 min 72o for 2 min          45 cycles  72o for 10 min  extension time  (Belgin Gocmen)  (this program worked best for Taxus and Douglas-fir)

Gels:

Reactions were run out on 1.5% agarose gels in 1X TBE for 3-6 h at 90 to 110 V.

Kreike's extraction method was used for these RAPD projects (no phenol extractions). This extraction method uses SDS for lysis. 0.5% tween 20 has been shown to counteract the negative effects on polymerase activity. DNA was resuspended in TE pH 8.0.

RNase (20ng per 1 ng DNA) treatment can increase amplification several-fold. It is imperative that the RNase added is DNase-free.

  PCR 10X buffer:                                                   500mM KCl                                                       3.73  g 100mM Tris pH 8.3                                               1.20  g bring up to 100mls; filter; autoclave; aliquot and store at -20oC.    dNTP mix:  Boehringer-Mannheim dATP, dCTP, dTTP and dGTP [100mM]  make 10mM stock solution      125 ul each dNTP      500 ul ddH20  bring up to 1000 ul volume.

Literature used to modify the various steps of this protocol.

Demeke, T. and Adams, R.P. 1992. The effects of plant polysaccharides and buffer addition on PCR. BioTechniques. vol. 12 No. 3 (332-334).

Kreike, J. 1990. Genetic analysis of forest tree populations: isolation of DNA from spruce and fir apices. Plant Molecular Biology 14 (877-879).

Pikaart, M.J. and Villeponteau, B. 1993. Suppression of PCR amplification by high levels of RNA. BioTechniques 14: 24-25.