How much ligase per ug of DNA? - (Jul/20/2007 )
I am inexperienced in molecular biology, thus will ask a very basic question for some of you: how many units of T4 ligase (NEB) do you use to ligate 1 ug of cut plasmid overnight in general? (Give me a range).
I understand it varies depending whether ends are blunt or sticky. If I have an insert with one sticky, one blunt ends (vector - too), how many units of ligase will I need? (Insert 800bp, vector - 7kb; have not decided how much of each to use per reaction).
1µg seems a lot to me. I would better go for 100ng.
Anyway, 2µl for this amount should be sufficent
One Weiss unit corresponds to 67 cohesive-end units as defined by New England Biolabs.
In 30 minutes at 16°C, 0.015 Weiss units of T4 DNA ligase should ligate 50% of the fragments derived from 5 micrograms of lambda DNA digested with Hind III. (J. Sambrook and D. W. Russell, 2001. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, ME.)
There are seven fragments generated by a Hind III-cut of lambda DNA (48,502 basepairs = MW 31,526,300) and 5 micrograms of Hind III-cut lambda DNA = 7.2 e 14 ends. 0.015 Weiss units or 1 NEB unit should ligate 3.6 e 14 ends in 30 minutes at 16°C .
I use 1 Weiss Unit/67 NEB Units for blunt ends and 0.1 Weiss Unit/7 NEB Units for sticky ends.
Typically, the total DNA concentration in a ligation reaction should be between 1-10 micrograms/mL (according to NEB). I routinely ligate a total of 10 to 20 ng of insert and vector in a 5 microliter reaction, which equals 2 to 4 micrograms/mL.
1ug seems to much for ligation. Normally I would use between 20-30ngs of vector.
And I would use 1ul of T4 DNA ligase from NEB.
I would vary the time of ligation for sticky and blunt end ligations.
sticky ends, 30-60 min, and for blunt end - 2hrs
If you use 30ng of vector and 10ng of insert, would give a ratio of 1:3 Should be fine.
One thing to add to tfitzwater's usual erudite exposition: Since the NEB T4 DNA ligase has a concentration of 400 cohesive end units per microliter, his 7 cohesive end units for sticky end ligation is a very small fraction of a microliter -- to pipet it, you must dilute the enzyme. There is evidence, further, that excess ligase is inhibitory. Note also the low concentration of DNA he uses, which is optimal for recircularization, as opposed to formation of linear concatamers.
Thanks to everybody, I get the idea now. I was just too afraid to loose DNA, that is why I started my cloning with 1ug; thought I will ligate that much.
NEB T4 DNA Ligase Storage/Dilution Buffer: 50 mM KCl, 10 mM Tris-HCl, pH 7.4, 1 mM DTT, 0.1 mM EDTA, 200 µg/ml BSA, 50 % enzyme grade glycerol. Chill the buffer on ice before diluting the ligase. A 7 unit/µL stock prepared in ligase storage buffer can be stored for 6 months at -20°C without loss of activity.
This is a good way to save some money in the lab.