IUBio

strange effect in digesting cDNA library

David L. Haviland, Ph.D. dhavilan at IMM2.IMM.UTH.TMC.EDU
Thu Mar 11 11:44:58 EST 1999


At 16:50 3/11/99 +0100, Burkhard Hassel wrote:
>Hi netters!!
>
>When I treated a cDNA (library) with XhoI, the sesult on the gel looked,
>as if the cDNA smear shifted to a HIGHER molecular wheight.
>cDNA 1st strand synth was performed using methylated dCTP, so XhoI (MBI,
>Fermentas) should cut only in the site provided with the
>oligo-dT-primer. The aim is an unidirectional cDNA library. Genomic DNA
>was not visible in the poly-A RNA used.
>Has anyone of you an idea?

Burkhard:

Hmmm.... this sounds like the Stratagene cDNA kit???  Yes?

I had two experiences with this system.  One was where the library had very
poor 32P-dCTP incorporation in the second strand and was abandonded when
the yeild after sizing in low melt agarose was too low to continue.  

The second was more akin to your experience.  I was trained by someone that
prefered to use MeHg.  So I included some.  Initially it seemed to help as
the yeild and incorporation was spectacular.    Then I ran it on a gel and
was initially impressed.  However, a milisecond later I realized that my
average size wasn't 1.5-2.0 kb but more like 3.5-4.5 kb with a tail
exending upwards or 12-15 kb.

I heard that the Stratascript was good but this was too good to be true.
After some discussion with tech-serv, we concluded that the RT must have
"hairpinned" somehow.   In any case we attempted to ligate the "cDNA" into
lamda arms and all we ended up with was a library ligated to itself with
left-over linkers.   We had a more accurate, and more success with the cDNA
system from InVitrogen.

As usual, no affiliation.

So I can 't help but wonder if you are also getting some sort of
hairpinning going on in your system?

David


=============================
 David L. Haviland, Ph.D.
 Asst. Prof. Immunology 
 University of Texas - Houston, H.S.C.
 Institute of Molecular Medicine  
 2121 W. Holcombe Blvd.  
 Houston, TX  77030 
 Internet:"dhavilan at imm2.imm.uth.tmc.edu" 
 Voice: 713.500.2413  FAX: 713.500.2424
 ------------------------------------------------------  
I try to take one day at a time but lately several days
have attacked me at once!
=============================



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