IGEM:PennState/2006/Ligation

=Ligation=

Pre

 * Estimate DNA concentration of restricted fragments.  Aim for a 3:1 insert:vector molar ratio (660 g/mole/base pair).
 * Where x is the desired concentration of insert, y is the desired concentration of vector, and n is the current ratio of their concentrations, a 3:1 ratio implies the relationship 3/n*y = x.
 * Additionally, the volumes of the substances is governed by the equation total volume = volX + volY + buffer. Usually the total volume we use for ligations is 40μL.

Ligation

 * 1) for 20 μL reaction volume, to eppendorf add:

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.


 * For overnight ligation, store at 16C (15 hr)