HIS-TAG LIGATION - Ligation of His-tag into the pIMS vector (Oct/20/2005 )
Hi all,
I plan to insert his-tag with custom built Spe1 site oligonucleotides (total lengh 31 bases) into a pIMS vector (size 6 kb) following vector digestion with Spe1 and CIP. I will phosphorylate the his-tag oligonucletides before ligation. My concern is how to calculate the vector- insert molar ratio given taht insert is so small. The promega protocol for phosphorylation of oligonucleotides using t4 PNK mention to use oligonucletides at 100pmol concentration which is the cause of my concern as I will end with very less concentration to be used in a ligation mix with usual 3:1 insert to vector ratio (the whole dna in the ligation mix would be picomole well below 100 nanograms). So please advise how to follow this up.
Thanks.
Subu
I plan to insert his-tag with custom built Spe1 site oligonucleotides (total lengh 31 bases) into a pIMS vector (size 6 kb) following vector digestion with Spe1 and CIP. I will phosphorylate the his-tag oligonucletides before ligation. My concern is how to calculate the vector- insert molar ratio given taht insert is so small. The promega protocol for phosphorylation of oligonucleotides using t4 PNK mention to use oligonucletides at 100pmol concentration which is the cause of my concern as I will end with very less concentration to be used in a ligation mix with usual 3:1 insert to vector ratio (the whole dna in the ligation mix would be picomole well below 100 nanograms). So please advise how to follow this up.
Thanks.
Subu
Well usually this type of very small linker is very effiently cloned the concern is to avoid the multiple integration of several of this oligos in a raw that might be the most tricky part ! As for the ratio I cannot give you best advice cause I hav'nt done that for many years !
They was a similar topic on this forum check the posts hereOligo Ligation
Hope it helps
Pesji
Yep -- I never bother to figure out insert:vector ratios anymore; I just take an educated guess and go for it. With a properly prepared vector and such a small insert, you'll clone it easily.