This protocol is used for screening from the 96-well split from your colony picks. You should have a very reliable PCR screen that has been optimized well ahead of time. Your PCR should be optimized to detect signal at 1 fg. A control vector can be prepared that can be used as a control for random integration, and 3-4 of these colonies can be picked to be used as controls for your PCR screen.
1. Centrifuge the v-bottom 96-well plates at 1000 rpms for10 min.
2. Remove supernatant, and add 40 ul of PCR Buffer/proteinase K solution (this should be freshly prepared ahead of time – see recipe below)
3. Seal plates and place in a warm humidified container (place damp paper towels in the bottom of the container) in a 55o C waterbath for 4 hours. Or use your PCR machine.
4. Centrifuge plates at 1000 rpms for 10 min. Transfer 5-10 ul for PCR assay (~1/5 of your volume for PCR). Inactivate the proteinase K by heating samples to 95o C for 20 min. Seal the rest of the plate and store samples at 4 C.
5. I do the 4 hour 55oC incubation and 20 min. 95o C inactivation in one long PCR program.
PCR Buffer/proteinase K solution:
67 mM Tris HCl, pH 8.8
16.6 mM Ammonium sulfate
6.7 mM Magnesium chloride
5 mM B-Mercaptoethanol
add 1 mg/ml proteinase K (from frozen stock of 10 mg/ml, store in small aliquots to minimize freeze/thaws)
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