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-   -   Help! Linker with phosphates??? (http://www.molecularstation.com/forum/dna-forum/84055-help-linker-phosphates.html)

florenciatell 11-16-2011 01:04 PM

Help! Linker with phosphates???
 
I would like to know in what situation I must use a linker with phosphates for making a ligation to a vector with T4 ligase. Becouse of all the DNAs have phosphates on the extremes, why I should ask for synthesize my oligo with phosphates?
Thanks !!!

pigfarmer 11-16-2011 05:52 PM

Re: Help! Linker with phosphates???
 
I assume you are going to PCR an insert and digest a vector to ligate it into? There is no need to get phospates on primers, you are undoubtably going to digest the PCR product anyways, or do some TOPO TA sort of cloning...

florenciatell 11-17-2011 01:32 PM

Re: Help! Linker with phosphates???
 
I want to ligate a linker of 27pb on a digested vector, that linker has the restriction sites of the ezymes that digested the vector, It must ligate! but I`m not having success with the ligation. That`s the reason I`m wandering if the linker need to have extra phosphates to allow ligation or is not nesesary.

Thanks.

pigfarmer 11-17-2011 08:44 PM

Re: Help! Linker with phosphates???
 
I'm not quite following, what is 27pb? And you did digest your insert, right?

mmorgan 11-20-2011 07:22 PM

Re: Help! Linker with phosphates???
 
Quote:

Originally Posted by florenciatell (Post 437192)
I would like to know in what situation I must use a linker with phosphates for making a ligation to a vector with T4 ligase. Becouse of all the DNAs have phosphates on the extremes, why I should ask for synthesize my oligo with phosphates?
Thanks !!!

If your vector is cut with 2 different enzymes, you don't need phospho-oligos, because the vector has 5' phosphates.

Instead of ordering the oligos with phosphates (which I think is a bit pricey) you can just do a kinase reaction (Polynucleotide Kinase) to put on 5' phosphates.

But as I said if you make oligos with overhangs that match your vector and use 2 different restricions sites, then you don't need the oligo to be phosphorylated.

sinan444 12-06-2011 09:18 PM

Re: Help! Linker with phosphates???
 
Thank you.

csxw 06-02-2012 01:33 AM

Re: Help! Linker with phosphates???
 
oh!!i think you must done,good luck.


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