Biotinylation of plasmid DNA

AAAACc aa374885 at OAK.CATS.OHIOU.EDU
Wed Mar 25 17:40:01 EST 1998



On 25 Mar 1998, J.S Good wrote:

> Hi
> 
> How do I attach biotin to plasmid DNA and to linear fragments of dsDNA and
> ssDNA? 
> 

Attachment of biotin to plasmid DNA


-cut your plasmid with enzyme 
-fill in 5' protusions using klenow+biotin 16-dUTP and appropriate dNTPs
 
Attachment of biotin to linear ds or ssDNAs

The mechanism of labelling is based on end labelling with
Polynucleotide kinase
-double or ss DNA should have OH group at 5'end
-use 1 nmol oligo
-5 microliter 10 PKBuffer
-10 microliter 1 nM ATPgamma-S in H20
-33 micolter sterile water
-1 microliter T4 PKinase (30 U/microliter)
incubate 37C for  1 hour

____dry solution under speed vac
----add 45 microliter 100 mM potassium phosphate buffer ph 8
----add 5 microliter N-iodoacetyl-N'-Biotinylhexylenediamine
----heat 50C for 1 hour
----purify biotyinylated oilgo with sephadex g-25


Good luck



> Any advice appreciated.
> 
> Cheers,
> 
> James.
> 
> 
> 
> 
> 
> 
> _____________________________
> James Good
> Department of Pathology
> University of Cambridge
> Tennis Court Road
> Cambridge CB2 1QP
> United Kingdom
> 
> jsg at mole.bio.cam.ac.uk
> 
> remove the capitals for email
> _____________________________
> 
> 




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