DNA Preparation and Digest Date _________________

Number of plates ________ Plate # Species

______ ________________________

______ ________________________

______ ________________________

______ ________________________

• Grow clones in a shaker at 37°C overnight in 1.2ml 2X YT + Cam (12.5mg/ml)
• Centrifuge at 2700 rpm for 30 minutes (Program #1)
• Decant supernatant and dry and place immediately on ice. Perform all following steps on ice
• Add 70ml GET/RNase buffer to each well (for 2x plates use 40ml GET + 300ml RnaseA (20mg/ml))
• Vortex until pellets are completely suspended
• Add an additional 130ml GET/RNase to each well and vortex briefly to mix
• To each well, add 200ml freshly made SDS/NaOH lysis reagent (for 2x 96-well trays use 35.2ml H₂O, 0.8ml 10N NaOH, 4ml 10% SDS)
• Cover with foil sealer and invert 20 times. Then place on side on a shaker for ~4 minutes, until the total time is 5 minutes
• Add 200ml KOAc to each well and rock ~10 times and place on ice for 10 minutes
• Centrifuge at 4000 rpm for 30 minutes at 4°C (Program #2)
• While centrifuging aliquot 300ml chilled 2-propanol to each well of a polyfiltronics receiver plate and tape the filter plate on top
• Transfer ~400ml of the supernatant being careful not to transfer any of the pellet
• Centrifuge at 4000rpm for 20 minutes (Program #3)
• Remove filter plate and discard supernatant
• Wash twice with 200ml 80% ethanol and dry DNA pellet in a Speed Vac
• Resuspend in 45ml TE and incubate for 30 minutes at 37°C
• Centrifuge at 3000 rpm for 5 minutes (Program #4)
• The following cocktail is prepared in a reaction tube on ice

Reagent 1 Reaction 1 Plate Total

10X Reaction Buffer 1ml 100ml _____ml

EcoRI 1ml 100ml _____ml

RNase (20mg/ml) 0.05ml 10ml _____ml

dH₂O 3ml 300ml _____ml

On ice, aliquot 5ml into each well

Add 5ml of the DNA prep to the cocktail and place cover on the plate

Incubate in PCR machine at 37°C for 2 hours

Add 2ml of 6X loading dye and load on to gel