request: inverse pcr protocol

Shahram Mori smori at nmsu.edu
Sun Jun 4 22:07:31 EST 1995


Klaus Eimert (klaus at sun.physbot.umu.se) wrote:
: Sorry, I forgot the subject line. Here it goes again:

: Dear netters,
: I am planning to use inverse pcr for cloning a specific 5' directly from
: polyA RNA. Can somebody provide an efficient protocol or point me to
: some recent literature, please.
: Thanx in advance!
: Regards

: Klaus

This is what I would do;
Go ahead and do a RT on the RNA. Then digest the DNA with an appropriate 
restriction enzyme. In order to ensure that you have had good digestion 
run an aliquote out on agarose gel.
Pheno/Chloroform extract and NaOAC ppt. Resuspend in TE. 
Dilute the DNA to about 1-10 ug/ml in ligase buffer and add T4DNA Ligase 
and incubate at 16C overnight. Phenol Extract and pptate and resuspend in TE.
Place in an appropriate restriction buffer that will give you a 
linearized product.
Amplify with 50pmoles of each primer and 0.2mM dNTP's, 2.5u of Taq. Make 
sure you choose the parameters of thermocycling correctly.
Cheers

--
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Shahram Mori
Program in Molecular Biology
Department of Chemistry and Biochemistry Box 3C
New Mexico State University
Las Cruces NM
88003
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