SSB13Ntbk - Mar21

Ligation of EcoRI/BamHI digests

• Set up the following reaction:

 6.5uL ddH2O
 1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
 1uL Vector digest
 1uL Insert digest
 0.5uL T4 DNA Ligase

• Pound upside down on the bench to mix
• Give it a quick spin to send it back to the bottom of the tube
• Incubate on the benchtop for 30min
• Put on ice and proceed to the transformation

Transformation by heat-shock

1. Thaw a 200 uL aliquot of cells on ice
2. Add 50 uL of water to the cells
3. Add 30 uL of KCM to the cells
4. Put your ligation mixture on ice, let cool a minute or two
5. Add 70 uL of the cell cocktail to the ligation, stir to mix
6. Let sit on ice for 10 min
7. Heat shock for 90 seconds at 42
8. Put back on ice for 1 min
9. Add 100uL of 2YT
10. Plate 70+ uL on selective antibiotics, let incubate at 37 degrees overnight