Help in Primer designing for expression...

shifalichatrath via methods%40net.bio.net (by shifalich from rediffmail.com)
Mon Jul 20 02:12:16 EST 2009


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
>Hi friends..
>
>I am working with pET vector system. for expression of the enzme in
>E.coli BL21.
>I have to design primer with Nco1 site in forward primer.
>so can anybody will suggest me about the placing of ATG site of
>ORF...whether it should be in the primer's R.E site or else again i
>have to add in the primer aftre the R.E site.
>
>waiting for suggetion from scientific community
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Shifali Chatrath
Graduate Student
Protein science Lab
Dept. of Biological sciences
National University of Singapore
Singapore
+65-65161210
+65-96393449


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