Must all BSP be nested PCR? - (May/18/2006 )

Hello,everyone. I am a newcomer of the Forum. Now, I have some questions.
1 For BSP, must I do the second round PCR?
2 I do the BSP using reference'primers, but I got no bands, I don't know why?

-swj-

QUOTE (swj @ May 18 2006, 11:27 PM)

Hello,everyone. I am a newcomer of the Forum. Now, I have some questions.
1 For BSP, must I do the second round PCR?
2 I do the BSP using reference'primers, but I got no bands, I don't know why?

Hi,swi
You donot have to do a second round PCR.What Taq DNA polymerase do you use?I suggest you should the hot start Taq.I just do a 30 cycles and get my bands.

linfuan006

-Jim006-

QUOTE (Jim006 @ May 18 2006, 07:25 PM)

QUOTE (swj @ May 18 2006, 11:27 PM)

Hello,everyone. I am a newcomer of the Forum. Now, I have some questions.
1 For BSP, must I do the second round PCR?
2 I do the BSP using reference'primers, but I got no bands, I don't know why?

Hi,swi
You donot have to do a second round PCR.What Taq DNA polymerase do you use?I suggest you should the hot start Taq.I just do a 30 cycles and get my bands.

linfuan006

Jim, Thank you for your help. I use the hotstar Taq polymerase from Takara. But I get no band. Can you supply your reaction system and amplification programme for me ?

-swj-

Try lowering your extension temperature to 65C and increasing your extension time by 1.3x or so. See pubmed 8628694.

-phage434-

Thank you, Phaqe. I got the paper, I will do experiment again according the article.
But ,yesterday, I got a strange result. After second PCR, I got a band in the sample well. Could you explain it for me?Thank you very much!

-swj-