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BLUNT CLONING With EcoNI


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#1 Abousy

Abousy

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Posted 04 May 2011 - 05:45 PM

Hi Guys,

Just hoping anyone has advice on improving a blunt end clone?

The vector is linearized with EcoNI and the fragment i am inserting has been digested out with bgl II on both sides of the fragment.

I have repeated the process multiple times, though in general this is the order.

1) Digest Vector and Fragment
2) Gel Purify/Extract.
3) T4 or T1 Polymerase Treatment
4) Dephosphorylation with Antarctic Phosphatase for Vector Only.
5) Purify
6) Ligate with Promega T4 DNA Ligase at 16*C ON.


Thank you in advance

Abousy

#2 Abousy

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Posted 05 May 2011 - 10:12 PM

EcoNI digests CCTNN^NNNAGG
in my case the N's are CT^CAC. This leaves a 1 bp 5' over hang of 'C'. I would like to ligase with a bgl II site which is AGATCT. I've considered designing a bgl II linker for my vector, however i am also uncertain if a linker will anneal to the single bp from EcoNI?




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