Differences between primers for real-time PCR and RT-PCR - (May/29/2010 )
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
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.
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??
jehane on May 29 2010, 09:36 PM said:
"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.
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?????????
yes, in principle this is done in the same way.
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.