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dscDNA synthesis suitable for Illumina Sequencing

dscDNA Illumina

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#1 sierraocean

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Posted 04 April 2013 - 12:20 PM

Greetings,

I have been using Thermo Scientifics Maxima H Minus cDNA double stranded synthesis kit to generate dscDNA suitable for Illumina Sequencing after purification. The kit states that it works for 0.5ng-5ug of total RNA. There is no PCR amplification step with this kit.
I checked my rRNA depleted sample concentration using the Ribogreen Assay and got an estimate of 300ng/ul. I used 9ul of this total RNA along with 10pmol of random hexamer primers to try and generate longer cDNA fragments. However, when I went to check the dscDNA on a 1% agrose gel with EtBr, I could not see any product. This RNA sample was stored in liquid nitrogen for 5months so I don't think it degraded over time. I'm going to check the integrity using the RNA picochip for the Bioanalyzer 2100 but doubt that it degraded. Any suggestions on how to improve the first strand rxn? How could I check the first strand synthesis? I also am using the High Sensitivity DNA chip from Agilent to check for low concentrations of dscDNA but am not sure if this has been tried by others (the website states that it can be used for fragments of dsDNA and tech support did not say the chip is NOT intended for this use so I assume it's okay to use it).

-Sierra

Edited by sierraocean, 04 April 2013 - 12:24 PM.


#2 sofja

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Posted 08 July 2014 - 11:59 PM

Hi Sierra

Did you succeed? I did 2 rxns with oDT and RH primers (10 pmol) and i see ~10 kb band for oDT and a smear, and a relatively long smear for RH on agarose 1%, but i can't clone none of them.







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