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Differences between primers for real-time PCR and RT-PCR


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6 replies to this topic

#1 jehane

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Posted 29 May 2010 - 10:32 AM

Hello All
This is the first time i work on real-time PCR ------------and i would to ask if i could use the same primers used for reverse transcriptase PCR?

or there a difference between the two primers in each experiment???

Best regards

#2 tea-test

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Posted 29 May 2010 - 11:04 AM

if the product is specific and not too large (< ~200 bp) you can try them although some people prefer to order real time PCR primers with a higher degree of purification compared to primers for conventional PCR. But this is maybe also dependent on your oligo supplier.
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#3 jehane

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Posted 29 May 2010 - 11:36 AM

what do u mean by

"higher degree of purification compared to primers for conventional PCR"??

i ask mainly about the sequence of the primer oligonucleotide-------- so how i choose purified sequence??

#4 hobglobin

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Posted 29 May 2010 - 11:47 AM

what do u mean by

"higher degree of purification compared to primers for conventional PCR"??


Higher degree of purification means removal of salts, by-products of the reaction (mostly too short oligos) and similar stuff.
For a standard PCR, the oligos are just "desalted", a method for higher purification is for example HPLC. Check the website of your supplier, there the different methods are described more in detail.
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#5 jehane

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Posted 29 May 2010 - 12:08 PM

i see that

but i ask about the sequence of the primer used

as for example for normal rt-pcr we search for mRNA sequence in GenBank and then design the primers

so is this the same for Real-Time-PCR?????????

Edited by jehane, 29 May 2010 - 12:22 PM.


#6 tea-test

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Posted 30 May 2010 - 05:48 AM

yes, in principle this is done in the same way.
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#7 Trof

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Posted 31 May 2010 - 02:22 AM

You can use the same primers for SYBR green assay as for RT-PCR, although it should be a small product (max 500 bp, 100-200 bp is best) and it's usually better if the primers are intron spanning, so that they don't amplify any residual DNA that may be present.

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