Get Prepared

You need to use 96-well plates with 2.2ml volumes (ref 410088 from Stratagene)

and the 'Wizard Genomic DNA purification Kit' (Promega, Cat. #A1125)

Let's do it

-Pellet cells from 500μL of overnight culture by centrifugation at 3000rpm for 5minutes
-Resuspend cells in 150μL of 50mM EDTA
-Add 10μL of Zymolyase 2mg/mL . Vortex
-Incubate for 30-60 minutes at 37°C
-Centrifuge at 3000rpm for 5 minutes
-Discard the supernatant (by aspiration)
-Add 150μL of Nuclei Lysis Solution and vortex
-Add 50μL of Protein Precipitation Solution and vortex 20’’
-Incubate on ice 5 minutes
-Centrifuge at 3000rpm for 10 minutes at 4°C
-Transfer the supernatant (200μL) in a new deep well plate
-Add 150μL of Isopropanol (Room Temp). Mix
-Centrifuge at 3000rpm for 30 minutes at 4°C
-Discard supernatant (by aspiration)
-Wash with 150μL of room temperature 70% Ethanol
-Centrifuge at 3000rpm for 10 minutes at 4°C
-Aspirate the ethanol and air dry the pellet
-Add 50μL DNA Rehydration Solution with 1μL of RNase.
-Incubate 15 minutes at 37°C
-Rehydrate at 65°C for 1 hour or overnight at 4°C (Δ evaporation, final volume around 20-25μL)

For PCR, take 1μL

Dernière mise à jour : ( 10-03-2008 )