PCR problem:inverted repeats

BCRUBIN at weizmann.weizmann.ac.il BCRUBIN at weizmann.weizmann.ac.il
Tue Jan 24 12:47:18 EST 1995


I used the Taq Extender kit (promega) for a stubborn region and got a nice 3kb
fragment.
  Eitan.
*******************************************************************************
Eitan Rubin the 3rd                                                           *
* Plant genetics                                                              *
* The weizmann Inst of Science                                                *
* Rehovot, Israel                                                             *
* Tel: 972-8-342421                                                           *
* Email: bcrubin at dapsas1.weizmann.ac.il                                       *
*                                                                             *
*******************************************************************************
In article <vam2-1401951327240001 at anatomy-mac-9.uchicago.edu>
vam2 at midway.uchicago.edu (Viraj A. Master) writes:

>In article <0098A66B.25CBE724 at vms.csd.mu.edu>, 5gf4srivasta at vms.csd.mu.edu
>wrote:
>
>> HI netters!
>>         Im trying to PCR a 3-4 Kb DNA fragment which contains 1.6 Kb of
>> inverted repeat sequence. I get a shorter product which would correspond
>> to a secondary DNA structure loop (DNA hybridizing to itself) hence the taq
>> is unable to read through the entire template. I have tried hot starts to
>> eliminate this but to no avail. Does any one out there have any suggestions?
>>
>>
>Hi Namrata,
>
>I have not faced this problem, but I was wondering if you had to use Taq,
>or could you use a more thermostable DNA pol. such as Vent or Pfu (I have
>used Vent exo+ at 99C denaturing temp to amplify a highly GC rich
>template).  If you used one of them, perhaps you could dentaure at a
>higher temp. for a longer time.  Maybe this would alleviate your secondary
>structure problems?
>
>Good luck,
>
>
>Viraj
>
>--
>Viraj Master
>Department of Organismal Biology and Anatomy
>University of Chicago



More information about the Methods mailing list