Enzymes to be purified

Gene Synthesis Error Correction

ErrASE Error Correction Kit (Novici Biotech) or CorrectASE from ThermoFisher.

Surveyor Mutation Detection Kit: Transgenomic.

T4 Endonuclease VII: Affymetrix.

Thermus aquaticus MutS DNA mismatch repair protein: Excellgen.

Endonuclease V, T7 Endonuclease I, and T7 DNA Ligase NEB

Ligases

Ref: https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0190062

T4 DNA ligase  has high activity on blunt- and 2-base overhangs, and low activity on 5′-single base overhangs. T3 DNA ligase ligates only blunt ends well; PBCV1 DNA ligase joins 3′-single base overhangs and 2-base overhangs effectively with little blunt or 5′- single base overhang activity; human ligase 3 has highest activity on blunt ends and 5′-single base overhangs.

ractions containing expressed proteins were applied to a HiTrap Heparin column (GE) and eluted with 20 mM Tris-HCl buffer pH 7.5, containing 10% glycerol, 1 mM DTT, 0.5 mM EDTA and a 50 mM– 1 M NaCl gradient. Purified enzymes were dialyzed into a storage buffer containing 10 mM Tris-HCl buffer, pH 7.5 containing 50 mM NaCl, 0.1 mM EDTA, 1 mM DTT, and 50% glycerol and stored at -20°C. For the PBCV1-N-term-ZnF DNA ligase an additional 200 mM NaCl was added to the storage buffer to promote enzyme stability.

1X T4 reaction buffer or NEBNext® Quick Ligation reaction buffer, 100–1000 nM ligase

PEG stimulates all the tested wild type ligases, with T4 DNA ligase showing the best activity on all of the end types (Fig 2). The remaining ligases all show activity on the two blunt-ended substrates, especially in the presence of PEG. T3 DNA ligase did not have significant activity on substrates with a single- or two-base overhang, even in PEG containing buffer (Fig 2B). However, PBCV1 DNA ligase showed activity on single base overhang substrates (SBO), displaying a preference for 3′SBO over 5′SBO while hLig3 showed a preference for 5′SBO over 3′SBO, and both showed the ability to seal the two- and four-base overhang cohesive ends (Fig 2C and 2D). It should be noted that hLig3 remained very tightly bound to the ligated DNA after reaction, necessitating the inclusion of a 30-minute proteinase K treatment in the presence of SDS to prevent gel shifts

2196 pT7A3E-PBCV-1-Lig, 

http://molbiol.edu.ru/eng/scripts/01_04.html

PBCV1 (SplintR) Ligase 1 µl ==>(25U)==>100~1000nM in 20 ul, for 1000 nM in 20 ul, will need 712 ng. So make 1 ug/ul, or 1 mg/ml stock

1000 units/25U==> 40 ul. need to aliquot 40 ul of 1 mg/ml to make 1000 units/vial of lot 157-2196

Muts

use