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PCR working, qPCR is *not*.


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#1 Jestr22

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Posted 09 October 2014 - 09:44 AM

I'm doing a protocol that includes the capture of sonicated DNA fragments (previously 1% formaldehyde-fixed) with a complimentary biotinylated oligonucleotide.  

 

After incubating with streptavidin-linked magnetic beads and enriching for my target sequence (and subsequent de-crosslinking), I can amplify that sequence very nicely with traditional, agarose-gel endpoint PCR.

 

However, if I use those same samples for qPCR (SYBR green) instead of traditional PCR, I see no amplification above background noise.

 

Is there any reason that PCR would work as expected, but qPCR would not?

 

Thanks!


Edited by Jestr22, 09 October 2014 - 10:02 AM.


#2 Tabaluga

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Posted 09 October 2014 - 12:11 PM

How does your qpcr melt-curve look like ? Did you check the product on gel ? Are you sure everything is correct with the qPCR reagents (Sybr Green etc.) ?


Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#3 phage434

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Posted 09 October 2014 - 01:30 PM

I would prepare a double-size sample, and run the identical sample in both instruments with the same cycling conditions. Your instruments may be failing, or you may have marginal conditions. Try extending the extension time slightly. Also, try lowering the annealing temperature slightly.



#4 Jestr22

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Posted 10 October 2014 - 06:17 AM

How does your qpcr melt-curve look like ? Did you check the product on gel ? Are you sure everything is correct with the qPCR reagents (Sybr Green etc.) ?

 

Melt curve looks good -- as you would expect.  I did not check the qPCR product on a gel -- but why would the qPCR product look any different on a gel than the traditional PCR product?  I'm not being combative, I would really like to know, because in theory they should be identical.

 

I would prepare a double-size sample, and run the identical sample in both instruments with the same cycling conditions. Your instruments may be failing, or you may have marginal conditions. Try extending the extension time slightly. Also, try lowering the annealing temperature slightly.

 

In this situation, what is the logic behind lowering the annealing temperature?  My instinct would be to increase the annealing temperature....



#5 Tabaluga

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Posted 10 October 2014 - 07:50 AM

of course it should not look different in theory, but in theory also the qPCR should give a product in the same way as the PCR which is evidently not the case here.
I'd also go for a lower annealing temp because if i'm correct a higher annealing temp increases specifity, but you have the opposite problem i.e. no amplification.

Il dort. Quoique le sort fût pour lui bien étrange,
Il vivait. Il mourut quand il n'eut plus son ange;
La chose simplement d'elle-même arriva,
Comme la nuit se fait lorsque le jour s'en va.

 


#6 Jestr22

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Posted 10 October 2014 - 07:58 AM

of course it should not look different in theory, but in theory also the qPCR should give a product in the same way as the PCR which is evidently not the case here.
I'd also go for a lower annealing temp because if i'm correct a higher annealing temp increases specifity, but you have the opposite problem i.e. no amplification.

 

I should clarify, there isn't no amplification at all.... there is no amplification above background noise.  The assay is weird, it is a new assay we're working on; I'm sorry for any confusion.



#7 phage434

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Posted 10 October 2014 - 08:45 AM

If your PCR reaction is barely working (as this one probably is) then minor changes in conditions can push it from working to not working. Annealing temperatures are one sensitive area. You are more likely to get a product (not necessarily the correct one) with lower annealing temperatures.






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