4 replies to this topic

[bear23456]

How to design a pair of universal tag in multiplex PCR?
The universal tag is about 20-24bp, attaching to the 5' of the specific primers, it's Tm is about 10℃ higher than the specific primers, after the first several amplication specific to the "specific primers", then heighten the Tm to suitable for amplication specific to the "universal tag".

Very thanks!

[HomeBrew]

I don't think you'll be able to add 20-24 bases and only have a 10℃ increase in Tm...

[bear23456]

HomeBrew, on Apr 28 2010, 05:10 PM, said:

I don't think you'll be able to add 20-24 bases and only have a 10℃ increase in Tm...

thanks, if don't consider temperature, how do it

[HomeBrew]

I am unclear on what you're trying to do.  What is the purpose of the "universal tag"?  In what way is it "universal"?  What role does this tag play?

[bear23456]

HomeBrew, on May 5 2010, 04:16 AM, said:

I am unclear on what you're trying to do.  What is the purpose of the "universal tag"?  In what way is it "universal"?  What role does this tag play?

  the "universal tag" is design to make the multiplex PCR's reaction easier. The multiplex PCR has two pairs of primer, one is the "universal tag"; the other is the "specific primer" which 5'-terminal is attached with universal tag. It's somelike the nest PCR. After first several amplification targeting at the "specific primer" which could generate the targeting region including the complementary of the "universal tag" pair primer. Then heighten the temperature to use the "universal tag" to amplify specific at the complementary of the "universal tag" . Thus to unify the multiplex PCR's reaction condition, and increase it's specific.

Very thanks!