PCR Fragment Problem

Michael Cooley szcooley at dale.ucdavis.edu
Tue Feb 7 12:15:02 EST 1995


hodges bradley l (segdoh at uxa.cso.uiuc.edu) wrote:
: Maybe you have amplified an alternatively spliced transcript that 
: contains both primewr annealing sites.  This is the exact same thing that 
: happened in our lab that resulted in the discovery of four alternatively 
: spliced transcripts of our gene.  Clone the smaller band and see what it is.

: On Sun, 5 Feb 1995, Luc Simon wrote:

: > In article <3gvf1n$p1q at ccshst05.cs.uoguelph.ca>, awright at uoguelph.ca
: > (Andre-Denis G Wright) wrote:
: > 
: > > 
: > > Dear Netters!!
: > > 	I am posting this for my lab mate who is having a bizzare PCR 
: > > problem. She is amplifying a 2.9 kb region and lately (with newly 
: > > synthesized primers) she is amplifying a 2.4 kb band along with her target 
: > > DNA. Even though the two bands were separated by 500 or so base pairs, she 
: > > carefully excised the larger 2.9 kb band from the agarose gel. After 
: > > the freeze-thaw method she extracted the DNA fragment only to find the 
: > > second 2.4 kb band present again. How could this happen? She has played 
: > > around with the annealing temp and even at 60 C, this second band is being
: > > amplified. What could be the problem. Any suggestions??
: > > 
: > > 
: > Dear Andre,
: > 
: > If you are confident that this is not a simple case of having a secondary
: > attachment site of one of the primers on the 2.9 kb template (AMPLIFY, the
: > software, might help to find that out if you have the complete sequence of
: > the template available), it looks like you have involuntary asymmetric PCR!
: > Try changing electrophoresis conditions, going from TBE to TAE for example,
: > and see if the "2.4 kb" changes its apparent size. If it does, it probably
: > is ssDNA, and you can probably just ignore it!!
: > 
: > Luc Simon
: > 
: > 

It is not at all unlikely to find a smaller fragment amplified from a gel 
isolation. What happens is that a very small quantity of that 2.4 kb band 
was produced in the original amplification (probably some non-specific 
amplification). During electrophoresis some of the smaller fragment is 
stuck in the gel and is isolated with the larger fragment. Now that it is 
amplified enough for you to see it, it is going to be very hard to 
remove. If you can go back to the original reaction, run it out in a gel, 
stick a toothpick in the gel, mix into a new reaction, and reamplify. But 
considering the size of the fragments, it probably won't help. Otherwise 
I would simply clone the larger fragment.


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