Hi everybody,
I would like to ask you, if you have some experiences with these kind of smears - greater than specific products, after dsRNA production. Our template (into in vitro transcription reaction) was purified pcr-products, lenght circa 400 bp with T7 promoter sequences, we used MEGAscript Kit (AM1330) - circa 1ug of template, folowing purification and precipitation with MEGAclear Kit (AM1908).
In the final results, we separated fragments (hoping we got only one specific band) by electrophoresis on 1% agarose gel. I am mostly affraid of too long in vitro transcription - 16 hours on 37°C - but is it even possible to produce so many non-specific fragments? More on, should we use different mass of template into in vitro transcription reaction, or separate final dsRNA on PAGE/denaturing agarose gels?
Have you got some idea, which would explain such results, please?
Thanks for your help.
Matej Medla
The in vitro transcribed (IVT) RNA is single-stranded. Does your dsRNA production is designed by the 400bp template contains sense and antisense sequences, and then let the IVT RNA pair to form a double-stranded during the IVT reaction? If so, these kinds of smears may be due to the incomplete double-strand formation. I suggest that you first denature the RNA completely by heating, and then slowly anneal it to form a complete dsRNA.
Kai Li yes, dsRNA is formed during IVT, then we heat samples on 75°C for 5 minutes (as it´s recommended in protocol) and bring back to RT (about 1 hour) and then we run agarose gel ELFO (non-denaturing). Finally we have these results.
Maybe (just an idea) we use 20 ng of plasmid DNA as an input to basic PCR with T7 primers - isn´t it too much? Should the plasmid DNA "contaminate" IVT, even if pcr-products are purified (before IVT)?
Matej Medla may be your template is too much, I usually used 0.1-1ng plasmid as template do PCR. but if you purified your PCR product before IVT, I think it should not affect the IVT reaction. I still think that results are caused by the process of annealing (incomplete) or running gel (if running buffer is too hot, it will cause denature of dsRNA, so run gel in an ice box is recommand).
Kai Li it´s true...I tried run PCR with T7 primers with only 5 pg of template - results were really better.
One simple solution to my problem (and everybody else who would got these smears, maybe Leiping Zeng ) put your samples into boiling water, 3 min and bring slowly to RT (I suggest just turn the heater off and wait). Important is to add EDTA (final conc minimal 1 mM) into your sample or use some elution buffer with added EDTA. Boiling water (100°C) will denature/separate your dsRNA and when you let it on RT it will hybridize...EDTA helps to hybridize your dsRNA specifically (higher stringency).
Smears should be some agregates which may be synthesized during IVT - it occurs sometimes.
Good luck!
Don't worry about the smear, it is normal, sometimes you may even notice an additional clear band having the double size of the desired one, this is normal too and even commented/discussed in the kit manual.
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