strange effect in digesting cDNA library

Burkhard Hassel b.hassel at dkfz-heidelberg.de
Fri Mar 12 04:11:55 EST 1999



David L. Haviland, Ph.D. wrote:

> Burkhard:
>
> Hmmm.... this sounds like the Stratagene cDNA kit???  Yes?
>

Hi, David!
No, no kit at all. First strand synthesis is with Gibco's Superscript II at 45
deg. Second strand was with an enzyme mixture of 100 u of E.coli DNA pol I and
3.2 u of RNase H according to the alternate protocol (unit 5.5 in curr. prot.
mol. bio.).


> 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.
>

2nd strand synth seemed to be optimal, the peak round 4 kb and up to >10 kb.

> 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
>

Sorry, but I don't know what you mean with "MeHg". Methylated GTP????

> 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!
> =============================

Can be. For the hairpinning, I think I will make another trial with a higher
temp of the 1st strand (perhaps 50 deg or more should work for superscriptII).

  Yours
Burkhard Hassel
Dep. Tumor Progression and Immune Defence
DKFZ (German Cancer Research Centre)
INF 280
D-69120 Heidelberg
Germany
fon 0049 6221 42 47 62
fax 0049 6221 42 47 60






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