Help with PCR reaction Phusion Flash - wrong sized fragment

L Celeste via methods%40net.bio.net (by celesteel from gmail.com)
Tue May 26 16:05:59 EST 2009


Hi everyone,

I am working on PCR amplification of a large fragment containing a gene with
an expected amplicon of 11 kb. We have been using Phusion Flash Master Mix
and been getting a bright 2 kb band and a few other faint non-specific
bands. We have sent the primers and the DNA template for sequencing. Since
there is a limitation on how large a fragment one could sequence, we only
know that each primer was able to correctly amplify the first 1 kb from both
end of the target 11 kb DNA.

We then use another set of primers (Let's call it B (+) & (-) )that bind to
areas near the middle of the 11 kb target fragment with the first set of
primers (Let's call it A (+) & (-) ) to see if we were able to amplife an
approximately 5.5 kb fragment. So with A (-) & B(+) primers, we got a clear
6 kb band. But with A (+) & B (-), we not only got the expected 6 kb band,
but also the often-seen 2 kb band and a few other non-specific bands. So, we
thought primer A (+) was not specific enough and tried to redesign the
primers, but the new A (+) primer still give the same result as before.

Could anyone please provide some advice on what to do next or what could
have gone wrong?

Some extra information:
I have also done a negative control, and there was no band.

Thanks
Celeste


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