HELP! CYTOKINE RIBOPROBES.

sl16s at cc.usu.edu sl16s at cc.usu.edu
Mon Nov 21 23:26:54 EST 1994


	I am doing norhtern blots  to see the RNA levels of murine peritoneal
macrophage producing cytokines (IL-1a, IL-6, TNF) and T-cell producing
cytokines (IL-2, IL-3, IFN-g).  Iam using 'RIBOPROBES'. All my cDNA's are in p
BSKSII+ plasmid.  All cDNA's are transcribed with T7 polymerase  except IL-3
(T3 poly.) after linearizing with appropriate restriction enzymes.  Iam able to
get excellent results with IL-1a, IL-6, TNF, and IFN-g (conditions are;  Hyb
temp is 550 C, Hyb buffer contains 50% farmamide, Washing is high stringent(650
C)- which are standerdized by me.) but I am getting lot of background with IL-3
and IL-2 probes. I tried different Hyb temperatures from 42 to 600 C  If I
increase the temperature Iam losing all signals. If I decrease the temperature
Iam getting non-specific signals (High stringent washing didn't work). Here I
observed an interesting correlation!.  The probes for which I am getting
background are 370(IL-3) and 400(IL-2) bp in lenght where as others range from
800-1230 bp's.   Iam also unable to strip the probe even for the wet membrane. 
	Anybody had this kind of strange problem with the hybridization of
short riboprobes?.  What is the best way to avoid the background?.  Should I 
use different kind of probes like random primed or nick translated DNA probes.
( I am kind addicted to riboprobes because they so precise and accurate).  And
finally how to strip the membranes to reprobe the same membrane?.
		Thanks in advance.
							Sincierly
							Ravi Dugyala.
 




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