request: inverse pcr protocol
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!
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
Amplify with 50pmoles of each primer and 0.2mM dNTP's, 2.5u of Taq. Make
sure you choose the parameters of thermocycling correctly.
C A N A D A
Program in Molecular Biology
Department of Chemistry and Biochemistry Box 3C
New Mexico State University
Las Cruces NM
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