Hi everyone!
I am currently doing In vitro transcription of two different RNAs, one is ~90bp size and another one ~200bp. I am using 2μg of DNA template, the reaction is kept for 3 hours at 37 degree. After the purification, I am getting a clear single band of ~90bp size RNA transcript, while there's no band for the ~200 bp RNA (agarose gel). I tried increasing the incubation time to 4.5 hours, but still the same issue. The primers looks okay to me. I have run the DNA template on gel, there's single band. Please suggest me how to troubleshoot.
Several things could be considered for troubleshooting:
Extending incubation time (overnight at 37C works well for smaller transcripts)
Double or triple "G" after your promoter can also improve efficiency
Increase the purity of your PCR product (column-based purification)
Diogo Figueiredo Thanks for your suggestions.
I have added "GGAGA" after the T7 promoter, as suggested by the Invitrogen Megascript manual.
The PCR purification was done by Qiagen kit (column based), checked the DNA template on agarose gel after the purification and the ratios in nanodrop were okay as well.
I havn't tried overnight incubation. Next time, will keep the reaction overnight. Thanks again!
did you check the sequence of your DNA to look for possible mistake in the T7 promoter sequence?
Pratyusha Thakur Did you figure this out? I have a very similar situation. 124 bp DNA is PCR amplified, then I use a QIAgen agarose gel extraction to get my exact product and purify ~95bp RNA by denaturing PAGE to prevent secondary structure formation, since I'm making a hairpin. This process is very common in my lab, using "GGG" after promoter, desalting the RNA product before T7 (I tried with and without this step), running O/N, followed by 1 hr DNase. Yet, still no bands at all for my sample but the control is still working and I know it's not running off due to the ladder. Concentration verified by nanodrop.
I was wondering if you were using a denaturing agarose? I've just recently heard about this.
Hi! Catrin Law
My issue with not getting any RNA after IVT has been resolved by increasing the amount of the template. Actually, one of the RNA was sRNA and another one was mRNA. I was having problem with the mRNA (may be because of its structure difference). I generally run RNA in 1% agarose gel (RNase free), just to check their size and bands (if smear seen, then we can not proceed anyway). I do further experiments by running denaturing UREA PAGE.
You can run your RNA in denaturing agarose. I did once. I am attaching a link for the protocol that I followed.
Preprint Preparation of Denaturing Agarose Gel for RNA Analysis v1
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Techniques
- Blotting Techniques
- Immunoblotting
- Dyes