RT-PCR using S1 Nuclease

Shahram Mori smori at nmsu.edu
Sun Mar 19 18:48:46 EST 1995

Petra M. Jakobs, Ph.D. (jakobsp at ohsu.edu) wrote:
: From: Philip S. Ruban  Sahaayar at ohsu.edu

: I am trying to do RT-PCR, using S1 nuclease. Basically, I first extract
: my RNA and throw the probe of my interest into the total RNA. Then cut
:  with S1 nuclease and
: amplyfy the hybrid, using PCR. 
: Could anyone know the protocol for this method?
: If so, please me kno as soon as you can.
: Thanks.

To 20uL cDNA reaction add;
100 uL S1 buffer.
5uL of S1 nuclease (1unit/uL)
incubate at 37C for 30 minutes.
add 5uL of 0.5M EDTA (PH 7.5)
add 0.6 uL 20% SDS.
Run on Sephadex G75 equlibrated with TES buffer to separate cDNA.
See Perball Page 552. ( A practical Guide to Molecular Cloning).
Shahram Mori					   _/\_
Program in Molecular Biology			  _\  /_
Dept. of chemistry and Biochemistry Box 3C	  \_  _/
NMSU  Las Cruces NM				    ||

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