PCR from genomic DNA

GEORGE XIAOXI OUYANG, ELECTRICAL ENGINEERING, PHONE 579-0970 ldeng at kean.ucs.mun.ca
Sun Apr 10 11:13:50 EST 1994


In article <199404061949.AA00341 at wugate.wustl.edu>, krishnan at BORCIM.WUSTL.EDU writes:
> 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
Hi! Maybe you can try to Heat (95c) your template with primer etc.(No taq 
is added) for 5 min and 
immediately chill on ice. Spin briefly. Then  add taq. PCR 95/45", 37C( or 40c
)/1', 72c/3.5' for 30 circles. Try 1.5mM MgCl2 first. If you can get the 
band of interest you can adjust the conditions to get rid of the non-
specific bands and to get a higher yield. If not try change  MgCl2 to 2.0, 
3.0, 4.0 and 5.0 to see any improvement. Good Luck! 
					Lang-tuo Deng



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