Set-up the reaction as follows:
DNA – Xul*
10X Cre Recombinase Reaction Buffer – 5ul
Cre Recombinase – 1 ul (1 unit*)
H20 – Xul (up to 50ul)
Incubate at 37°C for 30 minutes and then 70°C for 10 minutes.
* One unit is defined as the amount of enzyme necessary to produce maximal site-specific recombination of 0.25 μg pLox2+ control DNA in 30 minutes at 37°C in a total reaction volume of 50 μl. The pLOX DNA is a 3.65 kb piece of linearized DNA with one pLOX site. Depending on the substrate, you will need to extrapolate how many pLOX sites must be digested and then adjust the units of Cre Recombinase accordingly.
- Increasing the amount of Cre Recombinase in the reaction can inhibit recombination by forming loxP dependent Cre-DNA aggregates.
- Longer incubation times will not improve recombination. Instead, this will likely lead to higher molecular weight recombination products.
- For additional usage notes, please see the Cre Recombinase product page.