In article <48t6ie$fdf at yama.mcc.ac.uk>
hfakhour at fs1.ho.man.ac.uk (hfakhour) writes:
> Hi !
>> We recently changed from using a kit for reverse transcription into using GIBCO
> M-MLv reverse transcriptase. We have been trying to sort out a protocol for
> that, but so far no success. The primers we use are random hexamers, our
> starting material is total RNA (1-2 ug), we do not use any labelled dNTP. The
> resulting cDNA is used for PCR. Could anyone please suggest a succesful
I have got a protocol that worked , but I tested it only with 9 ug.
9 ug of total RNA in 6 ul
5 min 65 degrees, quench on ice.
+ 4 ul 5 * buffer (250 mM Tris pH 8.3 / 375 mM KCl / 15 mM MgCl2 / 50mM DTT)
This is actually the first-strand-buffer from the Gibco cDNA kit.
+ 1 ul dNTP, 10 mM
+ 2 ul DTT, 100 mM
+ 1 ul HPRI 28 u/ul (USB)
+ 5 ul Hexas (Primer pd(n)6 with 200 u/ml from Pharmacia, diluted 1:250 in
+ 1 ul Gibco M-MLv reverse transcriptase, 200 u/ul
10 min 20 degrees
60 min 37 degrees
5 min 95 degrees, refrigerate
(The times and temperatures on a thermocycler for going home earlier).
+ 180 ul TE, ready.
one to ten ul for PCR.
For long PCR products it would be better to take superscript II from Gibco.
Hope that helped.
Burkhard Hassel (bhassel at orion.rz.mdc-berlin.de)