Help in Primer designing for expression...

Dear Vasu!
If you are adding NcoI site in FP, When after digestion of your insert amplified from what so ever template, you can clone it into vector digested with NcoI. So, actually, your Nco I site will remain intact after ligation and you will have ATG and this will be a part of your primer. But, before you order primer, you should make sure that starting from ATG your construct is in correct reading frame. As, Nco I site is CCATGG (I think, i didn't cross check.)Therefore, add two extra nucleotides GG or GC also to maintain your frame and you will have extra MG before start of your actual protein sequence.
I hope this is what you asked! your question was not very clear.
All the best.

Shifali

On Mon, 20 Jul 2009 00:13:29 +0530 wrote

Shifali Chatrath
Graduate Student
Protein science Lab
Dept. of Biological sciences
National University of Singapore
Singapore
+65-65161210
+65-96393449