a large insert ligation - (Oct/05/2006 )
helping...I subcloned a 8kb cDNA into pCDNA3(5.4kb, vector) using BamHI & NotI to digest . Then what is the optimal ratio of the insert : vector? Is 3:1 alright? I'm afraid too much insert will be ligated itself.
what's your opinion?
Can the insert replicate in the host cell or give the host resistence to your chosen antibiotic/selection agent?
If not, it doesn't matter if the insert self ligates as it can not survive in the host, or give the host resistence to selection.
The theoretical optimum ratio is actually 1:1. In practise that doesn't really work to well. So increase the amount of insert to as high as you can, and try to keep the ligating (insert +vector)mix as concentrated as possible. This would lean the reaction towards intermolecule ligation, (ligation between vector and insert.)
Hi el nino,
You could try to use this formula to calculate the concentration © of insert and plasmmid properly:
C of plasmid= 10-20ng/ul reaction volume
C of insert = [ng plasmid/(nu of plasmid/nu of insert)]x (5 to 10)