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Validation of PCR primers/ probes


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#1 helencollins560

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Posted 13 July 2012 - 02:25 PM

I will be go vaildating my real time primers/probes before I use them on "real" samples.
I have been told to run the product on a PAGE gel, however, is it also possible to use an agarose gel?

If not does anyone have a good recipe for a PAGE gel.

I have primers/ probes for mouse tissue, however, i wondered how to go about finding out whether these could also be compatible with rat tissue or if not, what software i could use to design some.

many thanks!

#2 tea-test

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Posted 17 July 2012 - 12:11 AM

i'm doing it always with agarose gels. As real-time PCR products are usually quite small I recommend 3% agarose gels.
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#3 Adrian K

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Posted 17 July 2012 - 04:16 AM

I will be go vaildating my real time primers/probes before I use them on "real" samples.
I have been told to run the product on a PAGE gel, however, is it also possible to use an agarose gel?

If not does anyone have a good recipe for a PAGE gel.

I have primers/ probes for mouse tissue, however, i wondered how to go about finding out whether these could also be compatible with rat tissue or if not, what software i could use to design some.

many thanks!


I am unclear, you want to measure your real time amplicons or real time primers using PAGE?
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#4 Trof

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Posted 17 July 2012 - 07:16 AM

I have primers/ probes for mouse tissue, however, i wondered how to go about finding out whether these could also be compatible with rat tissue or if not, what software i could use to design some.


Use BLAST with species specific setting (Others database, then select rat as organism) to check if your primers and probe match rat. If they don't, it's nowadays better to get them designed from the company selling fluorescent probes, they usually do it for free with your order.
You can use online software like primer3 to design primer-probe set instead, but you should look up the rules for primer/probe design first, probe isn't a cheap toy to mess up.

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#5 helencollins560

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Posted 23 July 2012 - 02:36 PM

Dear Adrian K- i wanted to test my primers to see if they work i.e. give a product on a PAGE gel prior to RT-PCR experiments

#6 Adrian K

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Posted 24 July 2012 - 06:19 AM

i see, if your amplicon very small i.e. <70bp, PAGE is a better option.
Expecting the world to treat you fairly because you are a good person is like expecting the lion not to attack you because you are a vegetarian.

..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...

"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong

"It's all just DNA. Do it."---phage434

#7 helencollins560

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Posted 01 August 2012 - 01:48 PM

tea-test

what is the protocol/conditions you use when you perform this and which loading buffer and ladder do you use?
the product sizes i should get range from 63-150




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