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neb ligase - (Jul/14/2010 )

Hi all

I am using neb liagse for thee first time and i am unable to understand the unit it defines . The vial says 2,000,000 cohesive end units/ml but the units given is generally weiss unit.

As per instructions :
Room Temperature Ligation:
For convenience, ligations may be done at room temperature (20-25C). For cohesive (sticky) ends, use 1 l of T4 DNA Ligase in a 20 l reaction for 10 minutes. For blunt ends, use 1 l of T4 DNA Ligase in a 20 l reaction for 2 hours or 1 l high concentration T4 DNA Ligase for 10 minutes. Alternatively, NEB's Quick Ligation Kit (NEB #M2200S, <30 reactions> or NEB #M2200L, <150 reactions>) is uniquely formulated to ligate both blunt and cohesive (sticky) ends in 5 minutes at room temperature.

Here its using 1ul of ligase for sticky which means that 2000cohesive units per ul. Why so high??
I am not able to know what should be the dilution that is hould perform in order to get the correct units for sticky and blunt end .

Plz can anyone suggest me something.
Thanks in advance

-deespike-

deespike on Jul 14 2010, 12:53 PM said:

Hi all

I am using neb liagse for thee first time and i am unable to understand the unit it defines . The vial says 2,000,000 cohesive end units/ml but the units given is generally weiss unit.

As per instructions :
Room Temperature Ligation:
For convenience, ligations may be done at room temperature (20-25C). For cohesive (sticky) ends, use 1 l of T4 DNA Ligase in a 20 l reaction for 10 minutes. For blunt ends, use 1 l of T4 DNA Ligase in a 20 l reaction for 2 hours or 1 l high concentration T4 DNA Ligase for 10 minutes. Alternatively, NEB's Quick Ligation Kit (NEB #M2200S, <30 reactions> or NEB #M2200L, <150 reactions>) is uniquely formulated to ligate both blunt and cohesive (sticky) ends in 5 minutes at room temperature.

Here its using 1ul of ligase for sticky which means that 2000cohesive units per ul. Why so high??
I am not able to know what should be the dilution that is hould perform in order to get the correct units for sticky and blunt end .

Plz can anyone suggest me something.
Thanks in advance

1 ul of NEB ligase is equivalent to about 4 - 10 Weiss units. This amount works fine for both blunt and sticky end ligations even with short incubation times. At least for sticky-end ligations you can get away with using a lot less enzyme (0.1 U), especially if you increase the reaction time. But why bother!

When you look at "old methods" much less ligase was recommended (0-1 - 1 Weiss unit/20 ul reaction). This was because in those days the ligase was extracted from non-recombinant E.coli strains and was 1) very expensive and 2) was not terrible pure and often contained small amounts of nuclease. Thus small amounts were used to save money and minimize the amount of contaminants added to the ligase reaction.

These days the recombinant ligases are very cheap and highly pure. Thus the use of high amounts of enzyme will not cause a problem as long as the final glycerol concentration is below 5 % (1/10 volume of ligase).

Hope this helps.

-klinmed-

Thanks for your suggestions. i think i know how much to use now.

-deespike-