Project:Sex typing with amelogenin/results-20120212: Difference between revisions

Project:Sex typing with amelogenin/results-20120212 (view source)

1,030 bytes added , 14 February 2012

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 * Chances are we *haven't* ruined our old master mix, which is great (£)
 * We should aliquot our master mixes so we don't have to put them through any more thaw / freeze cycles.
+Basically I think we are screwing up the PCR stage: using the wrong temperatures, too-short timings, or whatever. Here is a paper on sex typing with amelogenin:
+http://library-resources.cqu.edu.au/JFS/PDF/vol_39/iss_6/JFS396941356.pdf
+The paragraph relevant to PCR states:
+Genomic DNA samples were amplified in a 100 µl reaction volume containing 0.2nM of each dNTP, 50 mM KCl, 10 mM Tris.HCl pH 8.3, 4.0 mM MgCl_2, 120 pmoles of each primer, and 2.5 U Taq polymerase. PCR was run for 30 cycles of 94 C for 1 min (denature), 65 C for 2 min (anneal), and 72 C for 3 min (extend). Following amplification, the PCR products were analyzed by electrophoresis on 1.2% agarose gels run with 1 X TBE (89 mM Tris borate, 89 mM boric acid, 2 mM EDTA) or 6% nonenaturing polyacrylamide gels run with 1 X TBE. The gels were stained with ethidium bromide and the results were visualized by fluorescence under ultraviolet light.
+So, we need to:
+* Run each PCR stage for longer (2 to 3 times as long)
+* Use a higher annealing temperature