Ligation

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Protocol

  1. Determine the amount of plasmid and insert to be used through the equation X ng plasmid/[(1 M) X bp] = X ng insert/[(X M) X bp]. It is best to have a pasmid to insert ratio between 1:4 and 1:10. Note: a useful website for this calculation is http://www.insilico.uni-duesseldorf.de/Lig_Input.html. Also, using 150 ng of vector is a good starting point.
  2. Add the following volumes to a round-lidded PCR tube:
Reagent
Volume (μL)
DNA

X of insert

X of plasmid

5x T4 DNA ligase buffer
4
T4 DNA ligase
1
ddH2O
up to 20 μL
Total
20

Note: Larger total volumes can be used; however, it is best to keep the total volume as small as possible, without going below 20 μL.

  • Gently mix by tapping the side of the tube.
  • Centrifuge the tube for 5 seconds at 4,500 xg
  • Leave tube at room temperature overnight.


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