Variable yields in T7 trascritpion

Virginia Dress Dress at biosci.arizona.edu
Tue Mar 8 13:33:27 EST 1994


In article <CMBHsC.4Jo at usenet.ucs.indiana.edu>,
jgraham at bronze.ucs.indiana.edu (the End) wrote:
> 
> 
> I am finding enormous variability in terms of yield of specific labeled RNA's
> from day to day in basic T7 transcriptions. I have compared various 
> buffers (80 mM Hepes, 40 mM TrisOac, 40 mM Tris-Cl) and juggled other
> components (BSA, NP-40, spermidine, Pyrophosphatase, RNAsin) and reaction 
> times (1h, 1.5h, 2 h) as well as nucleotide levels appropriate for 
> making internally labeled transcripts (50-200 uM limiting nucleotide
> and 0.5-3 mM of the other three).
> 
> Overall, a particular working setup and template make work well one time
> in four or five.
> 
> I believe that differnces may be related to low nucleotide levels, as 
> sythesizing cold RNA shows variability, but far less completely dismal
> yields.

Are you always using the same batch/lot of hot nucleotide? Are you
aliquoting

hot nucleotide or freeze-thawing each time?  Are you using DEPC-treated
water?
Same batch across runs?  Are you freeze-thawing the cold nucleotides?  Are
the
nucleotides ph'd to around 7.0?  How big is the transcript you are trying
to 
make?   Please e-mail me with answers or post again to newsgroup.
																																		Good luck,
																																						Virginia Dress
								                              Dress at biosci.arizona.edu
																																						Biochemistry
																																						U of AZ
																																						Tucson



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