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.