Subcloning dirty PCR products

rosas at mcclb0.med.nyu.edu rosas at mcclb0.med.nyu.edu
Wed Aug 24 23:10:15 EST 1994


HI, there!!!

During the last week I've been trying to subclone some PCR products I
obtained using primers containing internal restriction sites. Such
restriction sites are located at a distance of at least 5 nucleotides
away from the 5' end. However, I haven't been successful, and it
seems as if no matter what insert:vector ratio I use, the insert won't
ligate into the vector (I have already tried different ratios and none
has given even one colony on my LB-Ampicillin plates). I know for a
fact that there is nothing wrong with my transformation, as I do get
colonies with a positive control, and I don't expect self-ligation of
the vector to occur because it was double-digested using
non-complementary restriction enzymes. 
My main concern is about something interferring with the activity of
the restriction enzymes when digesting the PCR products. My concern
comes from the fact that my PCR product appears as a very dark band
surrounded by some background that extends above and below it creating
sort of a smearring. I have run into this kind of troubles before, and
it seems to me as if whenever I get this dirtiness I have troubles
subcloning the PCR product. 
Now, here come my questions:
1. What are the most likely causes for my PCR product not to get into
the vector??? Incomplete or non-digestion caused by some interferring
by-product??? Others???
2. Is it possible to get ride of such by-products without repeating
the PCR reaction (I'm short of primers), say by cutting my PCR product
out of a LM agarose gel???
If somebody out there has experienced similar troubles before and has
got over it, I'd like to know how they did it. I will really
appreciate very much any useful suggestion. I know it may take much
of your priceless time to answer me, but...you know better than I do
how satisfactory for yourself
 it is to offer somebody a hand, knowing
that the other one has no more than a few words to give you
in return...THANK YOU!!!.

German Rosas.
NYU Medical Center
Internet: ROSAS at MCCLB0.MED.NYU.EDU  MCCLB0- 0 IS ZERO!!!.

p.s. Does anyone have the whole sequence of the plasmid pRE4??? If
you do, I'd be glad to copy it (I need it to set some restriction
digestion analysis on some clones I have). 



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