3 replies to this topic

[saibo]

Hi all,

I have primers that give product size of 420bp. Can I use them for real-time PCR?

I usually hear that real-time pcr primes should give a product size of 75-150. What is the reason for that?

I'll appreciate any replies.

Thank you very much.

saibo

[expresson_help]

I think the product of 150 bp is set so that the amplification time can be kept to a minimum. Perhaps the signal to DNA amount is also more linear over this range. That said, I have amplified up to 500-600 bp using QPCR before (Qiagen SyBR Green kit) so it can be done.

[littlecell]

please visit
http://dorakmt.tripo...s/realtime.html

[saibo]

Thank you for all the answers.

According to the link from littlecell:

Quote

6. Maximum amplicon size should not exceed 400 bp (ideally 50-150 bases). Smaller amplicons give more consistent results because PCR is more efficient and more tolerant of reaction conditions (the short length requirement has nothing to do with the efficiency of 5' nuclease activity),