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  2. → Viewing Profile: Topics: Podge

Podge

Member Since 29 Jul 2010
Offline Last Active Nov 27 2012 02:35 AM
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Topics I've Started

qPCR: band in gel, but super high Cq?

12 November 2012 - 07:19 AM

Hey guys,

I'm running RT-qPCR for relative expression on tissues.

I ran some testsamples on with conventional PCR (cDNA 1:10) and looked at them on a gel. In comparison to my reference gene the bands look alright and I expected them to give me nice Cqs. Also used 4x the cDNA in the testrun, but the bands on the gel actually didn't look different.

Ran the lot of my samples in the qPCR then, but only get Cqs of 37-39.

I am using the Roche UPL system, so the amplicon is detected by a probe that is specific.

Any ideas what could be the problem?

Thanks!

RNeasy kit + DNAse digest from Promega

23 October 2012 - 03:13 AM

Hey,

I will extra some RNA using the RNeasy Lipid tissue mini kit. In the protocol they suggest an optional on-column DNA digest. I don't have the Qiagen stuff for the digest, but I have Promega RQ1 DNase.

My question now is if I can use the RQ1 for the on-column digest or if I should digest after the RNA extraction or even before (read this somewhere)?

Has anybody tried this?

Thanks!

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