I have used Plasmid DNA with a specific insert cloned in the Plasmid as a template for setting up the IVT reaction. Here, I have used both T3 and T7 RNA polymerases to synthesize the antisense and sense RNA strands respectively. The expected RNA bands with T3 and T7 would be around 237bp and 229bp respectively, but after the completion of the reaction when I ran the IVT product on 2% agarose gel, I noticed that the RNA band obtained upon transcription with T7 (~229bp) is running slower than the one obtained upon transcription with T3(~237bp). what could be the possible reason for this?
try to estimate the size of the T7 product and look at the sequence; may be you have a T7 termination sequence (run of >5T on the DNA template) or a strong secondary structure
Sorry I read a little bit too fast... T7 and T3 RNA polymerases can make double stranded RNA (it turns back when reaching the end of the template) and initiate at one end of the linearized template if the restriction enzyme used for linearization is leaving 3' overhangs.... (like Pst1)
did you denature your RNAs (in 80% desionized formamide) before loading on gel: slow migration could be du to secondary structures.... How are you sure that it is the T7 product that is too long and not the T3 that is too short? (RNA sizing is not very precise in general...)
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