In-gel low-melt agarose ligation

Vector, insert or both can be in 1% low melt agarose gel slice. These should be melted at 70C prior to use.

Prepare 2 tubes at 37C:

1.)

Vector gel slice: 2ul

Insert/annealed oligos: 2-5ul

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H20: 10ul

Add oligos last.

2.)

5x ligase buffer: 4ul

T4 ligase: 2ul

T4 polynucleotide kinase: 1ul (if required)

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H2O: 10ul

Take T4 ligase from freezer just prior to use, it is temp sensitive.

Mix the 2 tubes together. Incubate at RT for 3hrs, or 16C or 4C overnight. Use 1-2ul of this ligation reaction to transform competent cells.