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PCR not works time to time - (May/12/2006 )

Hi,
I am trying to get PCR products from a cDNA library of Schistosoma and surprisingly some times it works and some time it does not in the same conditions, machines, PCR program and DNA template and reagents, excepts enzyme, dNTPs and buffer lot, though its the same supplier and works fine with other PCRs. One or two time it gave me band of different size. When it gave me band of exact size (that has been sequenced also and confirmed that I have the right product) it is a single and sharp band.
Please try to help.
Thanks a lot.
Debalina

-Debalina-

QUOTE (Debalina @ May 13 2006, 02:34 PM)
Hi,
I am trying to get PCR products from a cDNA library of Schistosoma and surprisingly some times it works and some time it does not in the same conditions, machines, PCR program and DNA template and reagents, excepts enzyme, dNTPs and buffer lot, though its the same supplier and works fine with other PCRs. One or two time it gave me band of different size. When it gave me band of exact size (that has been sequenced also and confirmed that I have the right product) it is a single and sharp band.
Please try to help.
Thanks a lot.
Debalina


HI there,
cDNA library means gene expression where sometimes its expressed and someties its not. Did you make the library yourself? May be the gene is transient expression. mAybe contamination? Try to sequence the funny bands to see what it is.
Anyways, PCR is a funny thing. NOw you see it, now you don't.

Chris tongue.gif

-chris_sylim02-

Hi friends,
It isn't funny if your PCR is "now you see; now you don't!"
Firstly you need to make sure something:
1. your PCR machine is in good condition (service enginneer will be in great help).
2. get a new set of reagents (enzyme, buffer and dNTP)

when you said "I am trying to get PCR products from a cDNA library of Schistosoma", are you mean that you are doing PCR from a plasmid?

regards

-Hadrian-