T7 RNA polymerase termination signals?
bernard at elsie.nci.nih.gov
Tue Jun 4 00:06:19 EST 1996
Bottom line: What are the sequences of phage polymerase
Yes, I am still trying to get good in vitro transcription of
a particular sequence for use in RNase protection analysis.
Basically I can only really use one region and this is next
to a T7 promoter so I am fiddling with the conditions. The
transcript I get is mainly full length but the yield is awful.
I tried 15degC for 3 h. It was worse then 37degC for 1 h.
I tried the Maslak & Martin buffer and this may have improved
things but the salt/detergent in the buffer screw up the gel
purification of the probe (I am working on this).
I tried beefing up the amount of enzyme or the amount of
limiting nucleotide (32P-CTP) but this didn't really help.
I have yet to try T4g32 protein or its equivalent.
In the back of the Ambion MAXIscript guide their is a vague
reference to accidental incorporation of phage RNA polymerase
termination signals in a template being a reason for poor
yield. No information is given as to what these sequences
actually are. I am wondering if I have blundered into
one of these at the T7 end of my transcript.
Can some virally-enhanced person help a poor
toxicologist and give me a pointer to what a termination
signal for T7 polymerase (and SP6 and T3 if possible)
look like? Much obliged for any pointers.
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
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