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Restriction sites.

Restriction sites. - Protocols and Methods Forum

Restriction sites. - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 06-28-2006, 09:58 PM
Ezhil Subbian
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Default Restriction sites.



Hi,

I designed primers to add new restriction sites but forgot to add any "clamp" sequences at either end. I have a KpnI site at one end and a BamHI site at the other. I realise my mistake. I'm curious to see if anyone has had any succes with digesting inserts with the restirction site at the very end. Or if its a lost case and I should just start over with fresh primers.

Thanks
E





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  #2  
Old 06-29-2006, 02:43 AM
Aawara Chowdhury
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Default Restriction sites.

In <[Only registered users see links. ].net >,
Ezhil Subbian <[Only registered users see links. ]> wrote:


There are several things you can do to clone with your existing primers:

a) Phosphorylate the primers using T4 polynucleotide kinase. Then do the
PCR using an error-correcting thermostable polymerase (Vent/Pfu/Pwo etc.)
Purify the PCR product, and self-ligate it (37oC for 1 - 2 hours). This
ligates your product into dimers/trimers/tetramers etc. NOW, cut with
KpnI and BamHI. Because you're cutting a concatamer, their recognition
sites are no longer at the ends. Purify the cut product, and ligate it
with your cut vector.

b) As an alternative, if you use Taq for PCR, just clone the product into
any TA-cloning vector. Do a miniprep, and cut your insert out of your
new TA-vector.

Good luck!
AC
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