3 replies to this topic

[Rico]

Does anyone know how to add one restriction enzyme site into the DNA fragment? i know how to add one restriction site in the primer at the 5' or 3' end by designing primer, but i wanna how to add inside of the DNA sequence...

Thanks a lot, a lot...

[leelee]

Haven't done it myself, but perhaps SOE PCR would be worth looking at?

[Rico]

just got it. divide the DNA fragment into two parts, for each part design two pirmers. For the two pairs of the primers, the antisense primer of one pair and the sense primer of the other one use the same restriction enzyme site, then digest ,ligase...

thanks Leelee, maybe the SOE PCR also work...

[leelee]

That is exactly what I meant...

Edited:
Sorry, just re read your response and realised you meant that you will do the two reactions separately and then digest and ligate.....(talk about reading something and reading what you think it says rather than what it actually does haha!)

Edited by leelee, 30 November 2011 - 10:59 PM.