Problem with RT-PCR for gene rearrangement

LAU Tze Chin tclau at
Fri Feb 9 21:50:11 EST 1996

Dear netters,

I am having some problems with  nested RT-PCR detection of 
BCR-ABL gene rearragement- non-specific smeary bands appears and the 
expected bands never show up. I start off by sticking to typical nested 
rt-pcr protocols (use to work in other tests.). 
Some queries :

1.Which reaction is more critical in affecting the 
 final results : the 1st pcr or the 2nd nested one. 
 I guess Mg2+ may be one factor affecting. So, should
 I vary Mg2+ in the 1st or 2nd reaction first.

2. I have tried to use Superscript RT from Life Techology in
   place of M-MLV RT, with no apparent improvement.How does
   diff. RTs affect the final outcome?

3. I have read from BM pcr application manual that there is 
   a so call Kogan buffer which is claimed to be superior 
   to conventional buffer for rt-pcr. How magic is it? 
   Should I give it a try?
   The buffer composition is :
   2 x concentrate: 134 mM Tris.HCl,pH 8.8; 3 mM each of dNTP, 
                    13.4 mM MgCl2; 13.4 mM EDTA; 33.2 mM (NH4)2SO4;
                    20 % DMSO; 20 mM 2-mercaptoethanol; 340ug/ml BSA.

Thanks for any advice.

Cordially, Gene.

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