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Old 09-06-2011, 07:24 PM   #1
ScottC
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Location: Monash University, Melbourne, Australia.

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Default 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.
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Old 09-08-2011, 06:33 AM   #2
pmiguel
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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?

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Old 09-08-2011, 02:40 PM   #3
ScottC
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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.
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Old 09-09-2011, 09:09 AM   #4
pmiguel
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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.

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Old 09-10-2011, 02:38 AM   #5
ScottC
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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.
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