Digestion/Ligation/Assembly

Restriction Enzymes

A catalogue photograph of four identical Thermo Scientific screw-cap tubes standing in a row on white, their labels reading 10x Unique Buffer, 10x Buffer Tango, Restriction Enzyme and 50x Oligonucleotide. Only the yellow cap on the Buffer Tango tube tells any of them apart at a glance.
Looking down into a blue freezer tray at four white screw caps, each with an enzyme name written on it by hand: BsaI-HFv2, BseRI, 2X G.A. Master Mix and BsmBI-v2. Three of the four are sitting upside down relative to the reader.

T4 DNA Ligase

A catalogue photograph of the Invitrogen 5X T4 DNA Ligase Buffer product: a white and red carton reading 5X T4 DNA Ligase Buffer, store at minus 20 degrees Celsius, two by 1 millilitre, catalogue number 46300-018, with two blue-capped screw-cap tubes standing in front of it on a white reflective surface.
A photograph of two New England Biolabs screw-cap tubes lying side by side: T4 DNA Ligase, catalogue number M0202S, and 10X T4 DNA Ligase Buffer with 10 millimolar ATP. Both are white-capped, clear-bodied and the same shape and size as the Thermo and Invitrogen tubes on the previous slides.

Setting up a Ligation Reaction

Ligation of Purified, Digested DNAs

THIS IS AN EXAMPLE PROTOCOL. FOLLOW THE PROTOCOL ON YOUR INDIVIDUAL WORKSHEET

Set up the following reaction in a PCR tube, in the following order:

6.5 uL ddH20
1   uL T4 DNA Ligase Buffer
1   uL Vector digest
1   uL Insert digest
0.5 uL T4 DNA Ligase
  1. Pound upside down on the bench to mix thoroughly
  2. Give a quick spin to send it back to the bottom of the tube
  3. Run the appropriate PCR program on your individual worksheet

Example PCR program

Run the "T4ligation" program on the thermocycler:

25°C for 30 minutes
 4°C forever
The SpongeBob SquarePants time card: the words THREE HOURS LATER, followed by an ellipsis, hand-lettered in teal capitals across a squiggly orange and yellow ground.

(or later)

Questions?