By mistake I took concentrated RNA (5ug) for cDNA synthesis(20ul reaction mix) and cDNA synthesis didn't happen. When I ran 2ul of that cDNA (after diluting to 50ul) in a gel, I could see RNA bands which were sheared. Can I make cDNAs again from that solution? Has anybody tried like this?
Can someone please help me,,,,
How do you know that cDNA didn't happen . You need to run at least a PCR to check your product. Of course if your reverse transcription reaction product still contains RNA you can re do it. Only make sure you dilute your RNA solution first. Also I would recommend to check your RNAse H because it seems not working if you still see RNA bands after your RT reaction.
Hope you are using Total RNA and not poly A+ RNA int he reaction. I suggest taking the reaction and try to purify RNA using some mini RNA column, to eliminate all reaction components and rerun the reaction. If not, you can take required RNA conc fromt he CDNA reaction and run 2nd cDNA synthesis. There is high chance of sucesses as you can see intact RNA on gel. Good luck.
Thank you for your valuable suggestions,,,I actually did PCR for GAPDH primer using the diluted cDNA template,,and there was no amplification. And I was wondering, even though i added more,how is it possible not getting atleast a very faint band of GAPDH? Since I wanted to find out where the exact problem is lying, I took less amount (2ug) of this RNA and a positive RNA (from which I had made very good cDNA from 2ug, few months ago) and followed same protocol for making cDNA. But, again, positive control again gave very nice amplification for GAPDH, but not for the other one, which shows that there is some problem with the RNA sample itself. What would have inhibited the RTase action in the RNA sample?
So, basically you have 2 batches of total RNA stock. Did you isolate these two batches of RNA at different time of plant development? Some genes only express (with mRNA production) at a specific development time or at a specific tissue of the plant species. Without mRNA, you cannot generate the specific cDNA you want. What about GAPDH?
@Yuan, Yes, I have two batches of RNA,but isolated from cell line not plant.
Something must be wrong with your enzymes. Did you check that ?
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