Problem with RT-PCR for gene rearrangement
LAU Tze Chin
tclau at ha.org.hk
Fri Feb 9 21:50:11 EST 1996
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.
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