Smearing-when PCR product is used as template

tannamal at canr.cag.uconn.edu tannamal at canr.cag.uconn.edu
Thu Oct 9 11:36:08 EST 2003


Hello all,

i am having some problems with my PCR and i hope some of you 
might provide me some insight:

i have to amplify a 1200bp product from bacterial genomic DNA 
with primers designed from a closely related organism. the first 
round of PCR with 45 degree C gives a faint 'expected' band along 
many non specifics. when i used a 5ul of that PCR reaction as a 
template for my next PCR with 50 C annelaing -i got a good 
"expected brand' along with some non specifics. there was no 
problem.
	 i gel purified the  expected band and used the gel extracted 
DNA as template to reamplify it- that also amplified the expected 
band but there was a internal smaller band amplification.
	so, at this stage i cannot use my pcr product as such for TA 
cloning - i had to gel purify the band- so i scaled up the reaction to 
get more expected band.

then the trouble started- from then on- always when i try to amplify 
my1200 band from PCR product as template or gel cut band as 
template-, when i run my gel-the whole lane would be a big long 
smear along with some 1200bp amplification. this whole smear 
bothers me and this is happeneing only for some time now and  i 
used both taq polymerase and also stratagene Easy A high fidelity 
enzyme- the second enzyme is giving more smears

i  know this post is long- but i am desperate bcos i have tried many 
optimizations and a seemingly simple step is blocking the whole 
research.

i eagerly wait for your suggestions.
thanks for your time.

arasu


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