ligation problem and dephosphorylate matters.... - (May/12/2005 )

hi all..
i single digesting my vector with HindIII and that resulting compatible ends,rite?same as i did to my insert,i also single digested it with HindIII,resulting also compatible ends.so,is it so necessary for me to dephosphorlate those vector and insert or can i skip it?for ur information, if the insert is successfully ligated to the vector, the ligated plasmid will have 2 antibiotics gene for screening..
2nd question, do we have to insctivate our ligases enzyme before transform?i never heard such thing but came acrross in one of this forum and became confused due to the short info..
thanks..help please...

Hi

you have to desphosphorlate your vector it can help you to avoid selfligation and also to eliminated false positives colonies.
When you try to transform, use 1-2,5 ul of the ligation reaction but you don't need to inactivate ligase, instead of the manufacture's protocol said that you have to do it, but normally is not necessary.
Also, try to make a negative con trol, using you desphosphorilated vector without insert and with ligase, it gives you an idea of % of religated vector (no desphosphorilated).

When you are trying to subcloning cohesive ends you have two possible subclonins, in the rigtth direcction and in the wrong, then you need to check it by resctriction digestion.

Good luck!

Baobab

1-2,5 ul will do the transformation?i used to load 10 ul of ligation mix..???

yes, 1 - 2.5 uL is usually enough (depending on the sizes of the fragments).

You will want to keep some of the ligation mix in case your first transformation fails.