Endy:DNA ligation using T4 DNA ligase
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Ligation Mix
Example - 10ul mix
- 1.0 μL 10X T4 ligase buffer
- Make sure this buffer is completely melted and dissolved. Precipitate is DTT. Probably best to aliquot this buffer into smaller portions, to reduce the freeze/thaw cycles. In general, make sure the buffer still smells strongly like "wet dog" (Checking if the DTT is still good.)
- 0.5 μL T4 Ligase
- 6:1 Molar ratio of insert to vector (~10ng vector)
- Dilute with ddH2O to bring the total to 10μL
Calculating Insert and Vector Amounts
[math]\displaystyle{ \rm{Insert\ Mass} = 6\times\left[\frac{\rm{Insert\ Length}}{\rm{Vector\ Length}}\right]\times \rm{Vector\ Mass} }[/math]
Procedure
- Let the 10 μL solution sit at 22.5°C for 30 mins
- Denature the ligase at 65°C for 10min
- Dialyze for 20 minutes if electroporating
- Use disks shiny side up
- Store at -20°C