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Can RNase inactivation takes place at 65 Degree for 48 h, incubation.

RNase inactivation hybridization

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#1 abpatel2288



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Posted 28 November 2011 - 04:43 AM

I am trying to do cDNA-RNA hybridization (subtractive hybridization) at 65 degree for 48 hrs,
  • i am isolation RNA from two different tissue say liver and muscle.
  • than i am synthesizing 1st strand cDNA of liver RNA with oigo-dT (labeled), followed by addition of oligo A and finally purifying cDNA with column.
  • Than using (liver) cDNA and (muscle) RNA in 1:1, 1:3 and 1:3 ratio of cDNA and RNA i am proceeding for hybridization(in 1X SSPE and Denhardt hybridization buffer at 65 degree 48 hours.
  • On completion of above step i am adding streptavidin beads, so that labeled cDNA will bind to it, along with RNA which have hybridized to it.
  • than i am trying to concentrate UN-hybridized RNA from supernatant, but i am not getting RNA...

IT seems that RNA is degraded at 65 degree either due to RNase activity or due temperature...

Please suggest modifications to carry out such hybridization process..

Also tagged with one or more of these keywords: RNase, inactivation, hybridization

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