Help with cDNA

Martin Kennedy mkennedy at chmeds.ac.nz
Fri Feb 25 19:14:14 EST 1994


In article <199402221805.KAA13779 at net.bio.net>, MUNGO at UVVM.UVIC.CA writes:
> I am looking for some help, opinions, or comments on a number of issues
> relating to RNA isolation and the construction of cDNA libraries.
> 
> 3) Finally does anyone have a procedure for using PCR to make high SA P32
> labelled probes that can be used to screen libraries ?  We presently use a
> Mungo
-- 
We've been using the following protocol to make probes for library screening. 
You can probably use much less template DNA, and I've seen several protocols
where only 10 cycles are used, but we can't get consistent probes unless we use
more cycles:

Generating probes by PCR:


IF USING THE TECHNE, TURN THE WATER ON AT THE TAP!!!!!!!!!


1. 	Set up a PCR reaction as follows:

			x ul template DNA, approx 5ng
			1 ul  of each primer @ 10 uM
			1 ul  of 10x reaction buffer
			0.5 ul  of 25mM MgCl2 (may need to optimize)
			1 ul  of 10x minus dCTP mix (2mM each dATP,dGTP,TTP)
			3 ul  [alpha32P]-dCTP (30uCi total)
			MPW to 10 ul 


2.	Then add approx 0.3 ul  Taq polymerase, overlay with a drop of oil and
perform PCR with appropriate conditions.  For example:

			96 oC/1 minute
			60 oC/1 minute
			72 oC/1 minute		30 cycles



3.	Add 100 ul MPW, spin briefly and remove from under oil.  Put through
G-50 spin column and denature as for oligolabelled probes.


Cheers,

Martin

NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
NN NN  NN       Cytogenetic and Molecular Oncology Unit          ZZZ
NN  NN NN           Christchurch School of Medicine            ZZZ
NN   NNNN              Christchurch, New Zealand              ZZZZZZZ
	      Phone (64-3)364-0880  Fax (64-3)364-0750



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