Arkin:JCAProtocols LigTrans

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Protocols Page

Ligation

This procedure begins with gel-purified DNA fragments in a total volume of 8.5 uL. If this is cloning a PCR product into a vector, or something altogether different, the procedure still holds if you make the total volume of DNAs and water be 8.5 uL

Set up the following ligation reaction in order:

  • 8.5 uL DNA and water
  • 1 uL T4 Ligase Buffer (not to be confused with T4 Polymerase buffer)
  • 0.5 uL T4 DNA Ligase (not to be confused with T4 DNA polymerase, T4 RNA ligase, or T4 Polynucleotide kinase, which are also in the freezer)

Let it sit at room temperature for 30 minutes, then you can transform.

Transformation

You can also transform by electroporation, but usually you'll want to heat shock chemically competent cells. Competent DH10B and TG1 cells are stored as aliquots in the -80 freezer as a communal stock.

  1. Thaw a 200 uL aliquot of cells on ice
  2. Add 50 uL of water
  3. Add 30 uL of KCM
  4. Put your ligation mixture on ice, let cool a minute or two
  5. Add 90 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 (longer incubation may work better)
  8. Put back on ice for 1 min
  9. For ampicillin selection, you can plate immediately, otherwise:
  10. Add 100uL of 2YT, let shake in the 37 degree incubator for 1 hour
  11. Plate on selective antibiotics, let incubate overnight
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