Problem with Ligation - (Jun/22/2007 )
I am having a serious problem with ligation. Both my negative control (DH5 transformed with digested vector) and postive control (transformation with uncut vector) show expected results. But the ligation dose not work. I dephosphorylate the vector with CIP before ligation and I use Sac II and XbaI for digestion. I use around 2 ug of DNA for Restriction digestion for 2 hours and then I run it on the gel, followed by gel extraction of the insert and vector and set ligation using 1:3, 1:4, 1:7, mixtures of vector to insert concentration. I still do not see colonies in DH5 alpha. After talking to another post-doc in the lab, he suggested that the amount of insert DNA ( 2 ug) is very low and he suggested that I use around 10 ug of DNA for getting a good insert. Is this true? Can any body suggest some trouble shooting ideas for my problem??
dephosphorylation should be based according the mols (number of molecules) rather then mass (ng).
My rule of the tumb for dephosphorylation is
0.1 Units CIP dephosphorylates 1pmol plasmid within 60mins while incubating at 37 Celsius in 50ul reaction volume.
How large is your plasmid, how much CIP did you use and how long did you incubate it for?
Over dephosphorylation will result in the ends of your DNA fragment being damaged and thus made unligatable.
Another common problem area is the ligase enzyme and ligase buffer. Both components go bad easily. Neither likes freeze thaw cycles. Thus, has anybody in your lab ligated a plasmid recently. Ie could the communal T4 ligase have gone bad? Is your ligase buffer still good. Always remember to aliquote the ligase buffer.
Since your ends are not compatible, then worst comes to worst you could forget the dephosphorylation and directly ligate your cut vector with your insert.
P.S: What is the nature of your insert? If PCR product, did you remember to add enough skirt bp around the restriction site? All enzyme have a footprint, and will not work if the restriction site is surrounded by a few bp. (the number of bp depends on the restriction enzyme)
How big is your vector and what is the size of the insert in relation to it?
If your plasmid is 10kb, and your insert is 500bps, then if you digest 1ug, you will get like 50 ng of insert. You will have to digest 10ug of DNA to get 500ng of insert. This could be purified and after some loss in the columns you could use it for ligation.
How big is your vector and what is the size of the insert in relation to it?
If your plasmid is 10kb, and your insert is 500bps, then if you digest 1ug, you will get like 50 ng of insert. You will have to digest 10ug of DNA to get 500ng of insert. This could be purified and after some loss in the columns you could use it for ligation.
What if the plasmid is 3.6 kb and my insert is 100bp? What would be the ratio? i am having some problems in restriction digestion.
How big is your vector and what is the size of the insert in relation to it?
If your plasmid is 10kb, and your insert is 500bps, then if you digest 1ug, you will get like 50 ng of insert. You will have to digest 10ug of DNA to get 500ng of insert. This could be purified and after some loss in the columns you could use it for ligation.
What if the plasmid is 3.6 kb and my insert is 100bp? What would be the ratio? i am having some problems in restriction digestion.
if you digest 1ug of DNA, you will get 27 ng of insert, so you will have to use like 10-15 ug of DNA to get a decent conc. of insert.