HOT cDNA PROBE

Dom Spinella dspinella at chugaibio.com
Wed Aug 13 20:06:56 EST 1997


Qin Zong writes:
> I'm going to make some cDNA probes from mRNA by using BRL's Superscript
> kit. I'd like to make them as hot as possible. So I'm going to reduce
> the cold dCTP conc. in the dNTPs mixture (I'm going to us hot dCTP,
> 3,000 mCi/mmol; 10mCi/ml). Can anybody tell me how much cold dCTP you
> added into the reaction mixture when you make the high SA cDNA probe? 
> 
> Thank you very much.
> 
> Qin

Qin:

My recipe for preparing a ultra-hot total cDNA probe from 1 ug mRNA in a
volume of 50 ul is:

DEP Water				4.25 ul
1M Tris-HCl, pH 7.5		2.50 ul		(50mM final)
1M KCl					3.75 ul		(75 mM final)
0.1M MgCl2				1.5 ul		(3mM final)
1M DTT					1.0 ul		(20 mM final)
RNAse inhibitor 		1.0 ul		(optional)
10 mM ATP				1.0 ul		(200 uM final)
10 mM TTP				1.0 ul		(200 uM final)
10 mM dGTP				1.0 ul  	(200 uM final)
1mM	dCTP				1.0 ul		(20 uM final)
Random Hex primers		17.0 ul		(300 ug/ml final)
20 mCi/ml a(32P)-dCTP	12.5 ul		(5 mCi/ml final)
Reverse transcriptase	1.5 ul		(6000U/ml final)

As you can see I use twice the label you do and cut down the cold dCTP
concentration 10 fold from 200 uM to 20 uM.  Good luck.

-- D.G. Spinella



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