dig-RNA synthesis

hcorbett hcorbett at bisance.citi2.fr
Sun Mar 19 06:27:27 EST 1995


Would someone kindly hazard an explanation for the following? 

I can synthesize 35-S labelled probes from a given linearized plasmid
with high yield and good label incorporation. I try to make the same
probe with digoxygenin-labelled UTP and get a terrible yield (though
it is labelled and I can still use the tiny quantity and get good 
label with the cold probe).

I sequenced the insert and it is not strikingly rich in A/T. After
making sure the linearized plasmid was very free of phenol I marginally
increased the yield, but it is only 5-10% of what can be expected using
this protocol (other people in the lab do the identical procedure).

Thus, I am using far more reagents than I would like to obtain a given 
amount of RNA. Any ideas what is going on?

Thanks - Heather

hcorbett at garnet.berkeley.edu




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