Help with PCR reaction Phusion Flash - wrong sized fragment

Dwayne Taliaferro via methods%40net.bio.net (by taliaferroD from mail.nih.gov)
Tue May 26 17:18:17 EST 2009


There are three possible scenarios.  They're somewhat exotic and I'm sure
someone on here will suggest a more pragmatic cause/solution

1.  The 2kb fragment is nonspecific.  Not likely since you say the 2kb
fragment contains 1kb from each end of the target.

2.  It is likely you are amplifying a version of the gene with a deletion in
it.  Possible since it is a smaller amplicon, it would out compete the
larger product.  You may see the larger product if you increase the number
of cycles.

3. There's significant structure in the ends being amplified such that they
effectively "looping out" part of the sequence or stopping the polymerase.
These truncated products have enough overlap to amplify each other to
produce a 2kb product.  Don't think this is likely.  Sounds crazy.

What I would do: 
A. Sequence the 6kb products to be sure they are correct.

B. Excise the two 6kb fragments and use them in a PCR reaction to see if you
can generate the 11kB amplicon.

C. If B doesn't work, increase the cycles on the original PCR to see if the
11kb product shows up.  If it does, cut it out and use it in another
reaction.




On 5/26/09 5:05 PM, "L Celeste" <celesteel from gmail.com> wrote:

> 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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