I am trying to clone a ~2kb portion of a ssRNA viral genome. It has high GC% (~60%), but the worst part is the secondary structure. It has a lot of complementarity, so the folding models predict that it is basically folded in half.
I have tried one-step Qiagen master mix, two-step with Superscript III at 53degrees, DMSO, Q solution, increased RT temps, touchdown PCR, priming with random hexamers and a few different genome specific primers, melting RNA at 65 degrees before RT, etc. Some experiments indicate that the problem is the RT step; the PCR works fine.
Please share your tips. I'm running out of ideas. Thanks.
Mike,
Good to hear from you, but I am atraid this is completely outside my area of expertise.
Lee Hansen
To clarify a couple things:
1) I know the PCR is working because I have a plasmid control that I can amplify.
2) I can amplify a ~200bp region using the cDNA I've made (with random primers or gene-specific primer). But that same cDNA doesn't amplify the entire ~2kb, like I can from the plasmid.
3) The plasmid I'm referring to was a commercially available clone. But I am trying to clone a different genotype from a patient serum sample.
So, it appears that the cDNA I made is not very good quality. I will likely try using Superscript IV with a longer incubation as suggested. I may also try a drastic 95 degree incubation instead of the usual 65 degree prior to adding the RT mix. I have also ordered some different primers.
I had the same issue than you and I increased the initial amount of RNA used in the RT step. I tested both random primers vs oligoDT primers, and the oligoDT were way better each time while amplifying my genes (4kb and 2.2kb).
We also used the GXL prime star PCR kit, which is really good with difficult PCRs. The only factor to play with was the amount of cDNA to use,which makes your life easier. If you can, you should email the company and try to get a sample (Takara).
Good luck with the troubleshooting!
I had the same problem some years ago. It was particularly challenging, because I used to have tiny amounts of RNA template, with good to poor RINs.
After trying different approaches, this was my solution:
Using Transcriptor cDNA synthesis kit (roche) with Random Hex and Oligo dT, including a denaturation step before adding the RT reagents, and then RT at 65C.
It worked great.
You can also try this (or other) approach using Gene specific primers for the RT, but you have to be really sure that the primer design is correct
Hello,
Some thermostable RT are commercially available (Rocketscript...) so you can do your first strand synthesis at up to 70-75ºC... that should definitely help you. Also, I did not note major differences between Superscript iii and iv for GC rich RNAs... Hope that can help.
Good luck
Betaine. I use 2M betaine (final concentration) in both my RT and PCR and it makes a huge difference! I found it more effective than denaturing at a higher or longer temp. For RT I use Superscript IV.
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