Brown: Team Resistance/11 October 2008


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11 October 2008

  • PCR Purification was done of the S105 gene.
  • Restriction digest with Spe and EcoRI performed in order to perform a ligation with the Biobrick Vector.
  • Ligation done with S105 gene and Biobrick Vector.

Ligation Reaction Procedure

  1. Thaw T4 DNA ligase and 10x Buffer on ice.
  2. In a sterile tube add (in this specific order): 1. 8 microliters dd water 2. 1 microliter vector 3. 3 microliters insert 4. 1.5 microliter 10x Ligase Buffer 5. 1.5 microliter T4 DNA ligase
  3. 2:45 PM 2 Hour Benchtop Ligation (products are kept at room temperature between 2-24 hours). When performing a short incubation, the concentration of ligase should be increased. Normally 1 microliter of T4 DNA ligase is used. Here we are using 1.5 microliters. If not proceeding onto transformation after the incubation period is complete, the ligation reaction can be stopped by freezing at -20 deg C.
  • It is important to note here the 3:1 ratio used when combining the vector and insert for the ligation reaction. The ratio is in terms of molarity NOT volume. Depending on the size of the insert, the volume of insert used will vary. With a very small insert, the ratio of insert to vector might increase to around 10:1.
  • The following equation can be used: Insert mass in nanograms = 3 x [Insert length (bp) / Vector length (bp)] x Vector Mass in nanograms