IGEM:PennState/2006/Ligation

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Ligation

Time: 15 min

Reference: NEB T4 ligase technical bulletin http://www.neb.com/nebecomm/TechBulletinFiles/techbulletinM0202.pdf

Pre

  • Estimate DNA concentration of restricted fragments. Aim for a 3:1 insert:vector molar ratio.

Ligation

  1. for 20 μL reaction volume, to eppendorf add:
Stuff Volume (uL)
insert x
vector y
dH2O z
10X T4 ligase buffer 2
T4 ligase 0.5
Total 20

where x is usually 5-15 uL, y=[1,5] uL.

Also, always remember to do a negative control, which will be like the reaction above, but w/ no insert. Your transformation plates will then be an measure of any vector re-ligation/uncut background.

2. For overnight ligation, add ice to cold water in styrofoam container to bring temp to 12-14ºC. Incubate overnight (15 hr.). Alternatively, for quicker ligation, ligate at RT for 30 min. Proceed directly to transformation.

Cloning

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