I have isolated plasmid and then linearised it with re. Then I put in-vitro transcription of the linearised plasmid but not band of mRNA could be observed only the linearised plasmid was seen. After DNase treatment the plasmid was digested and nothing can be observed. The plasmid has T7 promoter and I have used T7 polymerase (NEB) at 37 degree centigrade. Both linearisation and in-vitro transcription was done in 10x RNA polymerase buffer (NEB). What would have gone wrong?
Well 2- Ladder
Well 3 - Complete plasmid
Well 4 - Linearised plasmid
Well 5 - After in-vitro transcription reaction
Well 6 - After DNaseI treatment
My template size is 36kb inclunding the vector. The estimated size is around 29kb.
It's been 20 years since I've done in vitro transcription and I'm not aware of new technological advances in this area, but 29 kbp seems huge to me. My first explanation was that there were traces of RNAse in your solution (which you just need to test by spiking one of your failed reactions with total RNAs and then seeing if they are still intact) but 29 kbp, that seems too big to me. T7 RNA polymerase probably does not have good enough processivity to synthesize enough RNA to be visible on an electrophoresis gel. Last explanation: you produced RNA but there are RNAses in your electrophoresis.
Thomas Guiraud Shall I proceed with transfection of RNA that I have??
29kbp ? That is huge and you are likely never going to get a full length in vitro transcript. Why so big? Also, you gel is unlikely to resolve the RNA and plasmid of that size, they could even be running together as a high MW band. What kind of protein are you studying? The size of your transcript makes little sense.
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