6 replies to this topic

[jehane]

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

[tea-test]

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.

[jehane]

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??

[hobglobin]

jehane, on May 29 2010, 09:36 PM, said:

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.

[jehane]

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.

[tea-test]

yes, in principle this is done in the same way.

[Trof]

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.