Riboprobes, Northerns, and high background

Gil kgilbert at cmp.hmc.psu.edu
Wed Nov 20 10:21:55 EST 1996


Greetings,

I have recently been using riboprobes for radioisotopic detection of
mRNA species on Northern blots, and consistently get high background in
most lanes.  Here are the conditions:  Sodium-phosphate hyb solution
(Church & Gilbert); 65-68¡C hybridization; ~1 million cpms probe per ml
hyb solution; washes are two 15 min washes in 2XSSPE/0.5%SDS @ 65¡C,
followed by two 15 min washes in 0.1XSSPE/1%SDS @65¡C.

Probes are prepared from linearized templates containing respective
bacteriophage RNA polymerase promoters. [32]P-UTP @ 800Ci/mmol specific
activity. After transcription, probes are phenol:chloroform extracted
and ETOH ppt.  cDNA templates range from 250 to 1100 bp in length.

I detect single bands on Northerns, but get high lane background.  Any
suggestions on ways to eliminate this high backgorund would be greatly
appreciated.  Thanks in advance.

 
KA Gilbert, Ph.D.
Dept. Cell and Mol Physiol
Penn State Univ, Col of Med
Hershey, PA 17033
e-mail: kgilbert at cmp.hmc.psu.edu



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