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first strand cDNA sythesis with high GC contents


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#1 anonymous

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Posted 09 September 2001 - 09:00 PM

I have a problem with my RT. The mRNA has a very high GC contents in its 5' terminus(about 75%).How to overcome the secondary structute of this mRNA in first strand cDNA sythesis. I need your help if you have solution or experience for this. Thank you in advance.YAN

#2 anonymous

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Posted 10 September 2001 - 09:00 PM

Try using methyl mercuric hydroxide to keep the RNA denatured during the RT reaction.

#3 anonymous

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Posted 12 September 2001 - 09:00 PM

You may have a hard time with the methylmercury method, since methylmercuric hydroxide is not longer commercially available and is very toxic. However, look back at a message from 14 Aug. 2000 in this forum under the same subject. One solution suggested there was to use 18 mM dGdC dinucleotide in the reaction. I have not tried it but it seems to me that should bind to G's and C's in your mRNA without blocking the binding site of your oligo-dT primer. The polymerase should be able to remove the dinucleotides during the polymerization process.

#4 anonymous

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Posted 12 September 2001 - 09:00 PM

I have encountered much the same problem as what you describe.I would not recommend the use of dimethyl mercury, especiallybecause it is incredibly toxic and absorbs easily through the skin. There are two solutions....clontech hasa kit called GC melt which works exceptionally well. I believethe kit is for traditional PCR but I have used the GC melt buffer with success in RT reactions. Second option is to use 5 or 10 percent (final concentration) of DMSO in the RT reaction. Bothwork quite nicely and allowed me to RT-PCR sequences that I was unable to otherwise. Good luck.

scott


#5 anonymous

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Posted 20 September 2001 - 09:00 PM

Try PCR X Enhancer buffer from Gibco - it works fantastic on GC rich templates for PCR and probably willwork for your RT-PCR. Try 1X or 2X concentrations 1st.

- ryan






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