David LePage's Gene Targeting Protocols

<< previous | next >>

Blotting, Probe And Hybridization

I won't go over everything ad-nauseum, just a few pointers.

-Be aware that the gels are large: I keep the slabs within the casting tray during preparation and aspirate the solutions off with a 4 L side arm flask hooked up to a sink vacuum.

-Don't even try to lift slabs out with your hands: always use the casting tray to lift them out and slide them off the tray onto your transfer rigs.

-I usually do two slabs per rig. Since they're big they need a large volume of buffer--at least a liter per rig.

-I generally block the probe with Cot1 DNA from Gibco-BRL (catalog number 188440-016). They have a clear protocol for using it for this purpose. Since blocking is so easy I don't think it's worth the risk of not doing it.

-A two day exposure is worth the extra day to see faint bands from slow growing wells.


<< previous | next >>