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Rt - cDNA Forum

Rt - cDNA Forum. Discuss complementary DNA synthesis, cDNA libraries, and cDNA cloning in this forum.


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  #1  
Old 12-10-2010, 02:47 AM
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Smile Rt



I am trying to do RT but some RNA secondary structure blocked me from getting the full length, is there any reverse transcriptase that works well in high temp. and low salt? any idea how to solve this? thanks
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Old 12-10-2010, 09:23 AM
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Default Re: Rt

I take it youve tried heating the RNA at 60 degrees for 10 or 15 mins before you do your RT? If not give it a go.

Rob
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  #3  
Old 12-10-2010, 09:51 AM
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Default Re: Rt

You could even heat it higher then just cool it before you add the Rt.
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Old 12-11-2010, 01:16 PM
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Default Re: Rt

I renatured my RNA with DNA primer at 95oC for 1.5mins, then I add the dNTPs, RT buffer. after that i incubate at 52oC for 5 mins before I add the enzyme. but the secondary structure is still there and barely see full length.
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Old 12-13-2010, 12:09 PM
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Default Re: Rt

If you have enough sample , Id say give it a good long heat at 75oC (20 mins). then start at 95oC step straight away... dont let it cool.

Sorry I dont have any other solutions for you.
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Old 12-30-2011, 10:50 AM
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Default Re: Rt

Sorry for the late response to this thread. But I am facing similar kind of problems. I have got the full length gene sequence for my gene. Its 2500bp long approx. I have done 3 RACE s and got the full length by matching overlapping regions. Now I want to get the full length as one fragment. For that I have designed 3 primers from 3' UTR and 3 from 5' UTR. When I am doing PCR with deep vent, I am getting big smears even after 2-3 nested PCR. I have heated the total RNA at 65 C for 5 min and then kept it at ice and added the rest of the components for RT. The cDNA was made by oligodT primer at 42 C. Please help me regarding this as I am not getting any clue. I have also done another set of RT with high temperature RT at 48 C with one of my gene specific primers. But in that too the same story is approaching.
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