My new kit have precipitates in sp6 transcription buffer, i dont know why they are there? I used linearised plasmid cut with BamHI ( restriction site present in my gene primer). But nanodrop reading for the SP6 transcription reaction was very low as compared for the parallel T7 reaction with the same plasmid cut with SalI. I don't know what happened, is this due to precipitated buffer or something else? I purified template after digestion and used 10 microgram of template for the invitro transcription. Any one have any idea why SP6 reaction failed??
I suggest warming up the buffer at 37 and vortex it well. Then, before you start the reaction, warm up all reagents (except the enzymes) to RT for 30 min, mix well and set up your reaction. I had the same problem with Ambion's kit and that was the solution for us. I would try that.
Dear Manish,
My experience with dsRNA preparation (with other kits) says that the efficiency of SP6 is slightly lesser than that of T7 RNA polymerase. However, it is not that less to demoralize you.
The SP6 buffer have higher MgCl2 concentration than that of T7 and both the buffers have spermidine. Considering both the facts I would recommend you to prepare the reaction mix at room temp, in case you are not doing it.
Plus, please check the RNA conc on gel instead of Nano drop. I, somehow, am not happy relying only on nanodrop.
Good Luck
Anil
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