RT-activity of Taq polymerase, HELP!!!!

Klaus.Matthaei at ANU.EDU.AU Klaus.Matthaei at ANU.EDU.AU
Thu Feb 16 16:53:25 EST 1995


>In article <1995Feb15.092756.15170 at news.unige.ch>, mike at medsun.unige.ch (Mike
>Morris) writes:
>
>>ARe you sure that you are not looking at DNA? Most RNA extraction
>>protocols (except for CsCl cushions) also produce DNA; indeed, some
>>kits we have tried are better for DNA than RNA!
>>  Mike Morris
>>---
>>*******************************************************************
>>Division of Medical Genetics       tel (Switzerland) (22) 702.56.94
>>CMU, University of Geneva          fax (Switzerland) (22) 702.57.06
>>Geneva, Switzerland                email mike at medsun.unige.ch
>>
>>
>>
>I agree.  We routinely set up RT-PCR reaction controls with no RT added.  If
>an
>appropriately-sized band appears, then that means you're amplifying genomic
>DNA.  Introns in the genomic DNA (if they are not too big to amplify)can of
>course increase your band size.  It is thus best to design RT-PCR primers in 2
>
>separate exons.
>--Rich Moldwi


I think that it is even better to design your primers not only in two
different exons but IN two exons i.e. ACROSS an intron.  In this way you
cannot amplify DNA since one half of you primer is in one exon and the
other half in another.

Cheers, Klaus

*************************************************************************
Klaus Matthaei
Head, Gene Targeting                            |           |
The John Curtin School of Medical Research      |  _--_|\   |
The Australian National University              | /      \  |
Snail mail: Canberra, ACT 0200, Australia       | \_.--._/<<|
E-mail: Klaus.Matthaei at anu.edu.au               |       v   |
Landline: 61 6 249 3782 fx: 61 6 249 0415
                        "Sometimes I'm the Louisville slugger"
                        "Sometimes I'm the Ball"
                                Dire Straits
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