SEQanswers

SEQanswers (http://seqanswers.com/forums/index.php)
-   Sample Prep / Library Generation (http://seqanswers.com/forums/forumdisplay.php?f=25)
-   -   TruSeq DNA adapters in RNA-seq prep... concentration? (http://seqanswers.com/forums/showthread.php?t=13911)

ScottC 09-06-2011 07:24 PM

TruSeq DNA adapters in RNA-seq prep... concentration?
 
Hi Everyone,

I'm in a situation whereby I need to use two extra adapters than the 6 adapters that are currently shipped with each set of the TruSeq RNA-seq prep kit and at the moment we only have one set. We have both sets of adapters for the TruSeq Genomic DNA prep kit (i.e. all 12 adapters).

I know the Genomic DNA and RNA-seq adapters are the same, but the concentration is different. Has anyone used the adapters from the DNA kit in the RNA sample prep? I think it used to be a 10 fold dilution of adapters using the 'old', non TruSeq kits.

Cheers,

Scott.

pmiguel 09-08-2011 06:33 AM

Can't you just check using absorbance at 260 nm (UV spec) if the UV spec or fluorescence (with oligreen) if UV spec is not sensitive enough?

--
Phillip

ScottC 09-08-2011 02:40 PM

Hi,

Yes, I've measured them with picogreen... I guess I should have phrased the questions slightly differently...

"Can anyone confirm that this will work, having actually switched the adapters after dilution and produced an experimentally tested library".

I expect that it will be fine, but whenever I modify the protocols I always like to check whether anyone else has already tried it before I blow a few thousand dollars finding out the hard way :-)

Cheers,

Scott.

pmiguel 09-09-2011 09:09 AM

I guess picogreen should not work well. It is a double stranded fluor that fluoresces very weakly in the presence of single stranded molecules. Hence it is not confounded much by RNA contamination of a DNA sample. For short oligonucleotides anneal over a short stretch I don't know you would get much signal.

But if your concentration results were fairly linear over a set of dilutions, I guess you will be okay.

Anyway, no, we have not tried it ourselves. Please let us know how it turns out.

--
Phillip

ScottC 09-10-2011 02:38 AM

Hi,

Yes, I realise PG detects primarily dsDNA, but I think the adapters have sufficiently large double stranded regions to work with picogreen. We really only need a relative concentration rather than an abolute concentration.

It appears that they're approximately 60 fold more dilute in the RNA-seq kit than in the genomic DNA kit.

We diluted the DNA adapters 1 in 50 and the preps appear to have worked well.

Cheers,

Scott.


All times are GMT -8. The time now is 09:42 PM.

Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2020, vBulletin Solutions, Inc.