Aim: Perform double strand cuts in a DNA sequence and ligate two DNA sequences together
Digestion
In a 1.5mL Eppendorf tube, add:
100ng DNA
1µL Enzyme 1
1µL Enzyme 2
2µL 10X appropriate buffer
Qsp 20µL ultrapure water
In a control tube, add the same components, but replace DNA with ultrapure water
Mix gently and incubate under agitation at 37°C for 1h
NB : Buffer to use
Enzyme 1
Enzyme 2
Buffer
SpeI
PstI
Cutsmart
XbaI
PstI
NEBuffer 3.1
EcoRI
PstI
NEBuffer 3.1
Purification
Gel purification : Run samples on electrophoresis gel and purify appropriate insert and vector.
See the appropriate protocol (Protocol 3 : Electrophoresis )
PCR purification : See the appropriate protocol (Protocol 8 : PCR and PCR purification)
Ligation
Add the following reaction in a microcentrifuge tube, respect the order (T4 DNA Ligase should be added last)
Molar ratios were calculated using NEB BioCalculator (http://nebiocalculator.neb.com/#!/ligation).
Molar ratio of 1:3 vector to insert shown
Components
20µL reaction
Nuclease free water
to 20µL
10X T4 DNA ligase buffer
2µL
Vector DNA
50ng (0.020 pmol)
Insert DNA
Xng (0.060 pmol)
T4 DNA Ligase
1µL
Gently mix the reaction by pipetting up and down and microcentrifuge briefly.
Incubate at 16°C overnight or room temperature for 1 hour
Chill on ice and transform 5μl of the ligation product into 50μl competent cells.
