PCR from genomic DNA

Mr. Steven Swift; BIOL; GRAD sswift at umbc.edu
Thu Apr 7 13:20:36 EST 1994


krishnan at BORCIM.WUSTL.EDU wrote:
: Hi!
: Could somebody tell me how to PCR-amplify a 3 kb piece of DNA from total human
: genomic DNA or from total yeast DNA (from a strain containing the respective
: YAC clone)?  I tried 1 microgram template, 50 pmol primers, 200 micromole dNTPS
: 2.5 mM Mg and 1 unit Amplitaq.  The cycling profile was: 96 C 2 min 1 cycle,
: 94 C 1 min, 55 C 1 min, 72 C 3 min for 30 cycles. I didn't get any product as
: determined by EtBr staining. Thanx in advance for any pointers.
: Raja
: krishnan at borcim.wustl.edu

	I've PCRed 2kb from yeast genomic DNA. My reaction was in 50ul.
  I used ~1ug template, 100umoles of primmers, 200umole dNTPs.
  Cycle ran - 
	Step 1 94C 4min
	Step 2 94C 1min, 50C 1min, 72C 2min 
		repeat Step 2 30 times
	Step 3 72C 10min

  Note that others in my lab have done larger sizes, they sometimes
  extend the elongations time greater than 1min per kb.
  ie. 3kb would be ~3min 30sec or 4 min.
  
  Also the Mg concentration sometimes has to be varied for best results.

  Good Luck

--


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