Help! Linker with phosphates???

[florenciatell]

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]

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]

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]

I'm not quite following, what is 27pb? And you did digest your insert, right?

[mmorgan]

Originally Posted by florenciatell

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]

Thank you.

[csxw]

oh!!i think you must done,good luck.