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DNA extraction and paraffin embedded tissue

PCR DNA extraction

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5 replies to this topic

#1 polymerase1986

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Posted 22 November 2011 - 09:35 AM

I’m trying to isolate DNA from a blood sample. Unfortunately the sample was not fresh, meaning the sample was already degraded. Any experience?
We’re also working with DNA from paraffin embedded tissue. So far we we're not able to amplify something (amplicon ~600bp). Do you have any suggestions besides increasing the cycle number?

Thanks for your help!!

#2 phage434

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Posted 22 November 2011 - 02:20 PM

Check out the PrePCR enzyme mix from NEB (similar products from Epicentre) that are intended to repair damaged DNA prior to PCR.

#3 nightingale

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Posted 23 November 2011 - 02:09 AM

regarding the FFPET samples, before going to the PCR.. check if u have DNA in the first place.
some paraffin samples contain PCR inhibitory dyes,,, unfortunately! Posted Image

Edited by nightingale, 23 November 2011 - 02:09 AM.

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#4 polymerase1986

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Posted 23 November 2011 - 11:26 AM

luckily there is DNA in the sample :-)

#5 nightingale

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Posted 24 November 2011 - 04:18 PM

that's good, is it stained ... ???
do u have another block with a different stain/color ???

have u run a housekeeping gene run ??? before going to test ur sample ???
maybe it's negative ... ???
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#6 Trof

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Posted 01 December 2011 - 03:14 AM

DNA in FFPE
- is fragmented though not to such extent as RNA, maybe try some small optimized amplicon on your samples
- is very reactive to formaldehyde so increased protease digestion (often overnight) is required to get good DNA

Edited by Trof, 01 December 2011 - 03:14 AM.

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