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How to set up real-time PCR for yes/no bands (rearrangement)


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4 replies to this topic

#1 ramblingrat

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Posted 16 June 2014 - 07:25 AM

Hi everyone,

 

I want to screen a large set of samples for presence or absence of bands and think there must be some way to do this by real-time PCR instead of running 100 gels.

Does anyone have experience with that and can recommend a good reference/source with instructions how to set it up?

 

Thanks very much!!

 

R.



#2 bob1

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Posted 16 June 2014 - 02:12 PM

HRM might be the way to go for this - it would work best if you have two different products that give different sizes based on presence/absence of the DNA of interest - as this way you would get two different peaks which would enable you to tell if 1) the PCR worked for that sample and 2) the presence/absence of the DNA of interest.



#3 Trof

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Posted 17 June 2014 - 02:15 AM

You said presence or absence of bands.

Not sure if you mean like two bands in one case and only one band in the other, or presence of one band in first case and absence of any bands in the other, but both would be possible to do even with SYBR chemistry only.

 

What is your actual set-up (band size(s))?


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#4 ramblingrat

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Posted 07 July 2014 - 02:11 PM

Hi,

 

I am looking for presence of one band in the positive case and absence of any bands in the negative. Meanwhile I figured out a qPCR that works with (expensive) SYBR mastermix. It also works as a normal PCR, however if I just add SYBR green I to the normal PCR reaction, the qPCR cycler doesn't really detect it (only when I load a gel). My amplicons are ~200bp. With another amplicon and SYBR green I, qPCR shows amplification, interestingly.

 

How does SYBR chemistry only work?

 

Thank you so much!!

 

R.



#5 Anh Nguyen

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Posted 03 August 2014 - 05:18 AM

There is just one answer: SYBR Green Master mix you used was optimized for short PCR products, just like your product 200 bp.

I think If your normal PCR has optimized, it will work if you add SYBR Green. Just be noted that you should used 0.5 X or 1X. not higher concentration. I might inactivate your reaction or lower PCR efficiency and lower sensitivity of the reaction.

Or else you should used EvaGreen instead. It always works to me. ^-^






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