Aug 08, 2017
Version 2
T4 DNA Ligase Reaction V.2
- New England Biolabs (NEB)Tech. support phone: +1(800)632-7799 email: info@neb.com
Protocol Citation: New England Biolabs: T4 DNA Ligase Reaction. protocols.io https://dx.doi.org/10.17504/protocols.io.irwcd7e
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: July 03, 2017
Last Modified: March 26, 2018
Protocol Integer ID: 6678
Abstract
This is the reaction for the "Ligation Protocol with T4 DNA Ligase"
Materials
STEP MATERIALS
T4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S
T4 DNA LigaseNew England BiolabsCatalog #M0202
T4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S
T4 DNA LigaseNew England BiolabsCatalog #M0202
Protocol materials
T4 DNA LigaseNew England BiolabsCatalog #M0202
T4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S
T4 DNA LigaseNew England BiolabsCatalog #M0202
T4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S
T4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S
T4 DNA LigaseNew England BiolabsCatalog #M0202
2 μl of T4 DNA Ligase Buffer (10X)
2 µL
T4 DNA LigaseNew England BiolabsCatalog #M0202
Vector DNA (4 kb) 50 ng (0.020 pmol)
50 ng
Note
Use NEBioCalculator to determine concentration
Note
recommended vector concentration 50ng=0.05ug
my linearized vector concentration= 5ug/ml=0.005ug/ul
1ul vector=0.005ug
how many ul of vector to make up 0.05ug?
(0.05ugx1ul)/0.005ug=10ul
thus, 10ul of vector should be added into ligation reaction.
Insert DNA (1 kb) 37.5 ng (0.060 pmol)
38 ng
Note
Use NEBioCalculator to determine concentration
Nuclease-free water to 20 μl
T4 DNA Ligase, 1 μl
1 µL
Note
T4 DNA Ligase - 100,000 units
T4 DNA Ligase - 20,000 unitsNew England BiolabsCatalog #M0202S