cDNA insert magically shrinks?!

Peter pemanuel at umabnet.ab.edu
Fri Apr 12 17:06:38 EST 1996


I put a 660 bp light chain of an antibody into a phage display vector (pComb3H).

Then I gel isolated it and ligated a 660 bp heavy chain PCR fragment in
the same way (Xho/Spe).  

Now a double digest with Xho and Spe makes the 2nd heavy chain appear to
be smaller than the light chain I put in first.

Prior to the ligation the heavy SEEMED to be the same size as the light
chain (660bp).

I'm thinking deletion of the heavy chain since it may be toxic.  BUT only
a small deletion? Does that happen this way.

Can the restriction enzymes cause the fragment to BE the same size but
APPEAR to be smaller than before.  

Perhaps a salt effect from the digestion mixture since there was no phenol
extraction before I ran this agarose gel.

Just concerned I may have had a deletion.

Any thoughts. Maybe the enzyme is stuck to the ends of the DNA.  I
remember that BamH1 can do that. right?

Peter

-- 
Peter Emanuel Ph.D.
University of Maryland
School of Medicine
410-706-429410-706-8012 FAX



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