Another RNA and PolyA polmerase

fzestabr at chip.ucdavis.edu fzestabr at chip.ucdavis.edu
Thu May 12 11:39:32 EST 1994


Dear Shahram Mori 

You wrote asking about

: adding oligo dT's or dA's to the 3'end of RNA?  : I am wondring if TdT
transferase can add poly dt's to the end of 3'OH'd RNA? :

We routinely add poly A tails to the 3' ends of RNA by using poly A 
polymerase. Since there was a similar post about the use of polyA 
polymerase following are the specific reaction conditions I use.


	RNA in 10 ul H2O
	Heat 80-90 C for 2 min
	Place directly on ice water bath to cool for about 10 minutes 
	Add 40 ul of rxn mix (below)
	37 C for 3 minutes (Yes 3 minutes)
	Stop with 2.5 ul 0.5 M EDTA pH 8.0

Then I usually ppt. for further use

Reaction mix
	25 ul 2X Buffer
	5 ul 10 mM ATP
	2.5 U polyA polmerase
	Water to 40 ul total volume

2X Buffer
	10 ul 1 M Tris pH 8.0
	 2 ul 1 M MgCl2
	10 ul 5 M NaCl
	10 ul 10 mg/ml BSA
	 2 ul 0.1 M DTT
	65 ul H2O
 		IMEDIATELY BEFORE USE ADD 1 ul 0.5 M MnCl2 to the 2x buffer.
		We found that the trick to a good poly A addition was
		adding the MnCl2 to the buffer fresh. 


I have used this for construction of a cDNA library and RT-PCR for RNAs 
which don't have polyA tails

Elizabeth Estabrook
(NMSU alumni)
emestabrook at ucdavis.edu



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