HELP!!! Having trouble with cDNAsynthesis! - (Aug/13/2001 )
Can anybody tell me how I can make cDNA from an mRNA which has a internal secondary structure with a melting point of 101.1 degrees cellcius (has a very high GC content)? I have used all the 5' RACE kits and they have yeilded nothing. I have looked at the old literature and have found that methylmercuric hydroxide can be used to totally denature RNA secondary structures, however, this is only used in denaturing gels. How can I use it with RT-PCR? does anybody out there have either a protocol for methylmercury or another that will work for me? I'd be most appreciative.
Secondary structures are formed by internal base-pairing as you might already know. This secondary structure has been successfully disrupted by us in the lab by including 18 mM of a dGdC dinucleotide in the reaction.
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