Hi
I am trying to do a pull down test so, in the first phase I should do invitro transcription for that I am using of T7 RNA polymerase from promega company. I designed a forward primer with T7 promotor. after extraction of DNA from the gel, I added under material;
1-template DNA 1µL
2- 5x transcription buffer 2µL
3- DTT 1µL
4- rNTP 1µL(Roche company) with biotin-U label
5-RNAase inhibitor
6-dH2O 4µL
7-T7 RNA polymerase 1µL(Promega company)
-----------------------
10µL
finally after mix with spin I incubated that in 37C for 3 hours but I did not see any band on acrylamide gel.
I repeated this protocol for many times and even I did it with linearized plasmid but it did not work.
I need one advise please.
Hi there,
Do you check the quality of your final DNA template both on gel and by UV spectra? How much of it do you put into the reaction mixture?
The following may be helpful:
https://openwetware.org/wiki/Sauer:In_vitro_transcription_with_T7_RNA_polymerase
Check the activity of your polymerase if it works fine with other templates and how long have you had it? Also consider putting the right amount of template and finally I would suggest you scale up to 20ul.
Best wishes.
thank you peter
I bought polymerase from promega recently so I think it works correctly. I put plasmid as a positive control aound 0.5ug in 10ul reaction. but again I did not have a RNA band on the gel with silver staining. i will scale up to 20ul. do you think this scaling up will solve my problem?
thanks again
Jacob O Peter
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