IGEM:Brown/Ligation Set Up
Setting Up the Ligation:
Determine how much DNA is needed. Usually start with about 2 ul of of backbone, which usually translates into about .2ug of DNA. A good rule for estimating DNA concentrations is that the limit of detection of a medium sized piece of DNA (about 5kb) on an agarose gel with 20 wells is about 50-100 ng, therefore 0.2 ug will be a thin but very clear band on a gel. Also remember that the larger the piece of DNA, the more ethidium bromide it will bind and therefore the brighter it will appear , so if a 10 kb piece and a 1 kb piece appear to have the same brightness the 1 kb piece actually has a 10x higher concentration of ethidium bromide as the 10 kb piece.
1. # of base pairs in your backbone DNA * 660 ug DNA/umole base pair= # of ug/umole for your backbone.
2. divide .2 ug of backbone DNA by the # of ug/umole for your backbone = micromoles of backbone you have
3. Calculate the number of ug of insert needed. Maniatis suggest using a 10 nM concentration of termini. In a 30 ul ligation this will be 3 * 10^-6 uMoles of insert.
4. # of base pairs in your insert * 660 ug/umole base pair=# of ug/umole for your insert.
5. # of ug/umole * 3* 10^-6 uMoles of insert = # of ug insert needed in your ligation. This should be about the same number as a 10 fold molar excess of insert over backbone.
If you do these calculations for a 10 kb backbone and a 1 kb insert, you will need .2 ug of backbone and 2 ug of insert in a 30 ul ligation reaction.
Set up the ligations as follows:
.2ug BB, 0ug Insert, 3ul 10X buffer, 0ul ligase, up to 30 ul sterile miliQ.
.2ug BB, 0ug Insert, 3ul 10X buffer, 1ul ligase.
BB + Insert:
.2ug BB, your calculation of ug for insert, 3ul 10X buffer, 1ul ligase.
0ug BB, your calculation of ug for insert, 3ul 10X buffer, 1ul ligase.
-Let Ligations go for 2 hours to overnight.
-Transform 20 ul into competent E. Coli (see transformation protocol.)