Ist strand cDNA purification.

Shahram Mori smori at nmsu.edu
Sat Jul 9 20:26:22 EST 1994


jr at dna.bio.warwick.ac.uk wrote:
: Dear all,

:  Does anyone know a good method to clean 1st strand cDNA from unincorporated 
: dNTP's and primers.  The Superscript protocol calls for use of a large amount 
: of primer to be present. Can I use GeneClean?

: Thanks in advance


: Jaz
This is what I Do, Jaz;
To my 30ul cDNA reaction I add 2ul of 6N NaOH			| RNA
Mix well and spin down.						|Hydrolysis
Incubate at 65C for 30 min.					| Step
Spin down the rxn mixture and add 2ul of 6N Acetic acid. Mix 	|
Add 80ul of 6M NaI and vortex for quite a while.		|

cDNA PURIFICATION STEP:

To the above add 8ul of Glass fines suspension and keep on ice with
occasional mixing for 20 minutes.		
Spin at 4C 12000 xg for 15 seconds.	
Remove sup and add 500ul of 80% ETOH and vortex Vigourously.
Centrifuge and remove sup. Do the last step again. and spin at 4C for 25
seconds.
Remove sup. SpeedVav dry the pellet for 2 minutes. Add TE or H2O, incubate
at 65C for 5 minutes. Spin at ROOM TEMP for 2 minutes. Isolate the sup and
 Precipitate the DNA with 3M NaAcetate and Ethanol like usual.
This cleans my stuff.
Cheers.
Shahram Mori
Program in Molecular Biology
Dept. of chemistry and Biochemistry Box 3C
NMSU  Las Cruces NM
88003

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-- 
Shahram Mori
Program in Molecular Biology
Dept. of chemistry and Biochemistry Box 3C
NMSU  Las Cruces NM



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