DNA Preparation

Recipes

Stock solutions:

Chloramphenicol (20mg/ml)

10N NaOH

10% SDS

0.5M EDTA (pH 8.0)

2.5M Tris-HCl

RNase A (10mg/ml)

Isopropanol

80% Ethanol

LB storage media Bacto Tryptone 10g

(1L) Bacto Yeast Extract 5g

NaCl 10g

Glycerol 75ml

dH₂O Q.S. to 1L

Chloramphenicol(20mg.ml) 1ml

Add about half the water to dissolve the ingredients in and then measure to the full volume with water. Sterilize by autoclave. When cooled to room temperature, add the chloramphenicol and mix in well. Aliquot 175ml of the media into each well of the 96-well plate (Corning 3799), requiring about 20ml total to fill each plate. We use the Q-fill2 (Genetix) automated dispensing system to fill all the plates in this procedure.

2X YT media Bacto Tryptone 16g

(1L) Bacto Yeast Extract 10g

NaCl 5g

dH₂O Q.S. to 1L

Chloramphenicol (20mg/ml) 624ml

Add about half the water to dissolve the ingredients in and then measure to the full volume with water. Sterilize by autoclave. When cooled to room temperature, add the chloramphenicol and mix in well. Aliquot 1.2ml of the media into each well of a 96-deep well plate (Beckman 140504) requiring about 150ml for each 96-well plate filled.

GET/RNase buffer Add Final concentration

Glucose 2.25g 50mM

2.5MTris-HCl (pH 8.0) 2.5ml 25mM

0.5M EDTA (pH 8.0) 5ml 10mM

dH₂O Q.S. to 250ml

Weigh out the glucose and add to about half the volume of water to dissolve. Add the Tris and EDTA. Bring the final volume up to 250ml with water and pass through a sterilizing filter (Corning 430767). Store this at 4°C. For preparation of enough buffer for 2x 96-well plates, you will need to measure out 40ml of this buffer. To the 40ml, add 300ml RnaseA (10mg/ml) immediately before use.

NaOH/SDS Lysis Buffer This needs to be freshly prepared from the stock solutions before each use. Add Final concentration

ddH₂O 35.2ml

10N NaOH 0.8ml 0.2N NaOH

10% SDS 4ml 1% SDS

Measure out the water and then add the stock solutions in a 50ml conical tube.

This will make 40ml total, enough for preparation of two plates. Do not chill this reagent or place on ice.

Potassium Acetate Buffer KC₂H₃O2 (F.W. 98.14) 73.16g

Glacial Acetic Acid 28.75ml

dH2O Q.S. to 250ml

Measure out the Potassium Acetate and dissolve in about 200ml dH₂O. Add the Acetic Acid and bring up to the final volume of 250ml with dH₂O. Pass through a pass through a sterilizing filter (Corning 430767). Store this at 4°C.

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Fill the necessary number of 96-deep well plates. For this we use an automatic dispenser Q-Fill (Genetix X3001) using the depp –well

From the streaked colonies grown overnight on petri plates, (80ml LB + 7.5% glycerol + 20 mg/ml) pick a single colony using a toothpick and transfer to 96-deep well plates filled with 1.2ml 2X YT+ 12.5mg/ml chloramphenicol and cover with (Qiagen 19571) and label clearly.

Shake in an incubator overnight at 300 r.p.m., 37°C

When done shaking place the 96-well plate in a centrifuge (Juoan GR442) using a swinging rotor for plates (Jouan 11175338 and 11174168) and spin at 2700 rpm for 30 minutes at 4°C. For centrifuging, we use an insert designed for this plate carrier (Jouan 85240761) wrapped with 2 sheets of paper towels for extra padding. This will help prevent cracking of the plates.

Decant supernatant and thoroughly dry pellet by inverting the plate and gently tapping on a paper towel. Place immediately on ice and perform the remaining steps on ice unless otherwise stated.

The pellets can either be wrapped in saran wrap and placed in a -80°C freezer for later preparation or placed on ice for subsequent DNA preparation. We use a big autoclave tray filled with ice for performing the following steps

For the dispensing the reagents in this procedure, we use reagent reservoirs (Pierce 15075) suitable for the 12-channel, 300ml pipetter (Rainin L12-300) used for delivery to the wells.

Make a suitable amount of GET/RNase buffer for the number of plates you have (see recipes)

Add 70ml GET/RNase buffer to each well

Vortex (VortexGenie2) the plates until the pellet is completely dissolved. This takes about 2-4 minutes if the pellets have been frozen andthawed, and about 6-8 minutes if not frozen.

Add an additional 130ml GET/ RNase buffer to each well

Briefly vortex once again to mix

Prepare the 0.2M NaOH/1% SDS solutions (see recipes) and add 200ml to each well. DO NOT place this reagent on ice or the SDS will precipitate out

Cover with foil sealer (Eppendorf 951-02-300-1) using a cover roller to make sure the cover is securely sealed

Start a timer for 5 minutes and invert 20 times

Place the plates on their side on an orbital shaker and shake until the 5 minutes is up. Do not allow more than 5 minutes total time from the start of inverting

Add 200ml chilled Potassium Acetate (see recipes)

Rock back and forth to about 45° for gentle mixing about 10 times.

Place on ice for 10 minutes

Centrifuge at 4000 rpm for 30 minutes at 4°C

While centrifuging, aliquot 300ml chilled isopropanol into a polyfiltronics receiver plate (Whatman#77011800)) Place a filter plate (Whatman 7700-1808) on top and secure with tape. Label the bottom plate.

When the samples are done spinning, transfer ~400ml of the supernatant to the to the top filter plate, being careful not to transfer any of the pellet

Centrifuge at 4000 rpm for 20 mintes at 4°C

Remove the top filter plate and decant off the isopropanol by inverting and then tapping lightly

Wash the DNA pellet twice by adding 2X 200ml 80% ethanol discarding the 200ml after each wash

Dry the plate as much as possible by inverting on a paper towel and tapping gently.

Place the plate on its side in a Speed Vac (Savant) on high for 10 minutes and dry the pellet completely

Resuspend the pellet by adding 45ml TE buffer (See recipes) and seal the top with a plate sealer (Fisher 14-245-192B)

Place in an incubator at 37°C for 30 minutes

Centrifuge at 3000 rpm for 5 minutes at 4°C

Transfer the 45ml to a 96 well plate clearly labeled with a lid