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Amplification of region from sperm RNA


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#1 oms

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Posted 28 December 2010 - 08:10 AM

I have this weird problem, i take swimup of the human spermatozoa. Then extract RNA using Trizol method, i get a very low yield, somewhere around 22ug/ml. This I use it as an initial template and amplify the product of around 1.9kb. When my RNA is Fresh i get a very faint band in the gel. But after a weeks time if I try to amplify from the same batch of RNA I dont get the band. This hs been consistently happening and very disappointing. Currently I was storing my RNA @ -20deg. What would be the problem? can anyone tell me. I am using a Onestep RT PCR Kit containing AMV RT and Hi-Fidelity Taq Polymerase.

#2 kalpanak

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Posted 29 December 2010 - 04:03 AM

RNA is more stable at -70. Try storing at -70. Avoid multiple freeze-thaws. hope this was helpful

#3 kajmak

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Posted 31 December 2010 - 12:06 AM

Do two steps. Use any reverse transcriptase and DNA polymerase that works for you.
And cDNA is better for storage than RNA.

#4 kajmak

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Posted 31 December 2010 - 12:08 AM

Another thing, if you know the sequence of the region you want to amplify, design specific reverse primer, instead of polyT.

#5 oms

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Posted 03 January 2011 - 09:08 PM

Another thing, if you know the sequence of the region you want to amplify, design specific reverse primer, instead of polyT.


Thanks . I think storing at -70 helping me out, and even avoiding repeat freeze thaws. Thanks




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