Hi !!
Has anyone tried cloning a microRNA in expression plasmid ? i am trying to clone the premature sequence of the microRNA of interest in pCDNA3.1. i use genomic dna as template. i am not able to get it cloned. i rechecked my primers as well. it will be really helpful if anyone who succeeded can share their ideas.
Thanks in advance
Roshni.
Hi Roshini!
I Xavier, I have done my PhD in microRNA-based therapeutics. I have cloned some microRNAs in different expression plasmids so I have some experience in that issue. As far as I understood your problem you are having problems to get a PCR product (1), to clone it in the cloning vector(2), or to detect the expression of the cloned miRNA?
My suggestions are the following:
(1) Try the troubleshooting of regular PCR (MgCl, Tm, elongation times...). If you get desperate just order new primers.
(2) Try making changes in vector and insert rations, check efficiency of the competent cells...
(3) Double check promoter, insert size, its orientation, polyA...
Contact me in case you need more details or suggestions!
Xavier
Article MiR-148a-and miR-216a-regulated Oncolytic Adenoviruses Targe...
Article Late-phase miRNA-controlled oncolytic adenovirus for selecti...
Dear Xavier,
Thank you for the kind reply. i have sent a message to you regarding my protocol and problems.
Hi..........
We use to clone Genomic premiRNA sequence with flanking ~200 bp on either sides in pTRIPz vector by PCR cloning..............Most of the times it worked for us
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biomolecules
- Nucleic Acids
- RNA
- Small RNA
- microRNA