Genomic DNA PCR trouble

Julie McCollum julie at sorggenome.tamu.edu
Tue Jan 28 11:53:22 EST 2003


We have been having trouble PCR amplifying from grass (sorghum) genomic
DNA (~750Mbp). Products ~1200-1400 bp are not amplifying.  Failed
results are either a blank lane (no amplification at all) or a lane
with smaller (<500 bp) products that occasionally form a ladder (there
is no sign of the larger expected product in these lanes).  We usually
don't have trouble amplifying 700-800 bp products. We are using Oligo
to design our primers, and we sequenced BACs to design the primers.  We
check the quality of the sequence before ordering primers. 

The DNA was purified with QbioGene's FastPrep system and has
successfully been used in other PCR reactions.

A typical 10 µL reaction consists of the following:
5 - 10 ng genomic DNA
5 pmol each primer
1 µL 10X PCR buffer
2 mM MgCl2
200 µM dNTPs
0.7 µL (3.5 U) AmpliTaq Gold (Perkin Elmer)

Cycling conditions:
10 min at 94 deg, 
followed by 33 cycles of:
1 min at 94 deg
1 min at annealing(55-60 deg) - temp predicted by Oligo
1 - 2 min at 72 deg
Followed by 30 min at 72 deg.

Does anyone have any suggestions?  Is there a better TAQ for this? 
Should we be using more template and primer, or alter our conditions in
some other way?

TIA,

Julie 


--
Julie McCollum
USDA-ARS, SPARC, CGRU
College Station, TX
julie at sorggenome.tamu.edu



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