Ligation

From FreeBio

{Note: The following protocol uses the Rapid DNA Ligation Kit to ligate DNA inserts into vector backbones. The kit can be found in the -20C freezer in rm 5096.}

1. In a 1.5ml (Eppendorf) tube, mix:
   • 2ul vector
   • 6ul insert
     {Note: As control, use 6ul ddH₂O instead.}
   • 2ul DNA dilution buffer (Tube #2)
     {Be sure to have thoroughly re-mixed the buffer in its tube 1st and then to have thoroughly mixed it in the rxn.} --dna1112 10:21, 6 Jul 2005 (EDT)
   • 10ul T4 DNA ligase buffer (Tube #1)
   • 1ul T4 DNA ligase (Tube #3)
     {Note: Minimize time enzymes are spent outside of freezer.}
2. Briefly vortex. Briefly spin down in a picofuge.
3. Incubate @ room temperature for 15min. If transforming, start thawing Top10 cells, SOC media, and pUC19 DNA.
4. If -80 door is stuck, wedge right foot against bottom of refrigerator and pull door handle with left hand.
   {Or, you can wait a minute or so as pressure equalizes.} --dna1112 02:10, 23 Jun 2005 (EDT)
5. Proceed to Bacteria Transformation or store in -20C freezer.