low yeild fo RNA after invitro transcription - (Aug/11/2010 )
Iam getting very low yeild after invitro trascription using T7. I have checked the purity of the DNA, and has been completely linearised. Followed the protocol, did ethanol purification before adding the polymerase and then added the required contents and kept in 37 deg of 2 hr for one and 4 hr for another, and did Lithium Cl ppption and quantified. The yeild was around 77ng/ ul. I dont know why I get a very low yeild. Needed help to over come this problem. Any help will be appreciated.
How long is your target transcript?
Have you tried a control reaction with the same batch of T7 RNA pol and reagents, but a well-characterized template?
The transcript is about 2.1 Kb in length and the DNA are fresh, may be a day or two old.
The concern is not that the DNA is bad, but the enzyme may be. A control as suggested above would help to determine that.