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How to do cloning with NcoI RE in pACYCDuet vector

Nco1 pACYCDuet

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#1 nanhannada

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Posted 20 December 2011 - 10:31 PM

Hi.
I have a problem with expression of a protein which has second residue exactly what I want to express.
I need to exrpress Mat-Lys~ and Mat-Asn with pAcycDuet vector which has His tag in MSC.
Because I want to delete His tag, I have no choice except Nco1 for RE.
Also. it is very important to express Lys or Asn as a seond residue in my protein.
Here is a problem!
Nco1 has ATG start codon in the restriction site and is followed G.
I mean Nco1 site is CCATGG, so I can not express Lys( AAA) or Asn(AAT) as a second residue after Mat because of G after ATG in Ncoi site.
What should I do? plz help me.
Have a good day~^^

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#2 phage434

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Posted 21 December 2011 - 06:19 AM

Ignore the cloning site of your vector and design PCR primers for amplifying the vector only, with 5' overhangs containing whatever restriction sites you need. Don't forget some extra 5' bases to allow cutting.




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