Ultrapure ligase from Enzymatics

[Aero]

Hi,

I am curious if anyone has tried the "ultrapure ligase" (as in the Quail et al, Nature Methods paper) from Enzymatics. Thanks!

A large genome center's improvements to the Illumina sequencing system.
Quail ME, Kozarewa I, Smith F, Scally A, Stephens PJ, Durbin R, Swerdlow H, Turner DJ
Nature Methods 2008; 5(12): 1005-10

[peromhc]

anyone.. wondering if it really makes much difference versus "regular" ligase.

[shurjo]

I use it and it works great for me (I can't make a comparison with regular ligase as I've used this as my sole ligase). What I can tell you is you get 400ul of the enzyme for ~$300, so it's not outrageously expensive.

[captainentropy]

anyone.. wondering if it really makes much difference versus "regular" ligase.

so if you look at the details between the two SKUs the only difference is the concentration of the ligase and the "rapid" includes a buffer that has PEG in it (for molecular crowding).

http://www.enzymatics.com/product_l603-lc-l.htm

http://www.enzymatics.com/product_t4-dna-ligase-hc.htm

The higher concentration is preferred for blunt-end ligations (since it's a lower efficiency reaction). However, for library construction we are ligating T-overhang adapters to A-overhang DNA fragments. The question this begs is why would we want to increase the efficiency of ligation between DNA fragments that don't have an A-overhang? And if it's correct (according to the Quail et al. paper) that the ligases from companies other than Enzymatics, have enough nuclease activity to convert some of the A-overhang fragments back into blunt end fragments then shouldn't we never use the rapid ligases?

Maybe someone has a cogent answer but I'm thinking I'm not going to use the "Quick/Rapid" ligases anymore (or just dilute them).

[ECO]

The higher concentration is preferred for blunt-end ligations (since it's a lower efficiency reaction).

This is a common misconception. Blunt ligation is extremely efficient. In fact 1T:1A ligation is the trough in the efficiency plot (lower than both blunt and >1 base overhangs).

[Isequencestuff]

We've tried Enzymatics' "ultra-pure ligase". Compared to a lot of other ligases on the market there does seem to be a significant increase in ligated fragments as determined by qPCR pre and post ligation ( their ligase is what we currently use now). Utilizing Covaris for shearing also seemed to increase our ligation efficiencies.

[Purification2013]

I am in the purification department for enzymatics. Company purity standards are ten fold others. Nuclease activity is near nil.