384-well DNA Sequencing Template Picking, Growth and Isolation using the Q-Pix Colony Picker, HiGro Oxygenated Shaker Incubator, Zymark Mini-Staccato, and the Velocity 11 VPrep

S.P. Lin, H. Jia, F. Z. Najar, D. D. White and B. A. Roe
Version 1.01 - updated 03-03-03

After transformation, as described in our lab protocol book:

  1. Add 1 ml of fresh 2xTY (or YENB) to each sample and recover the transformed XL1Blue (MRF') cells by incubating at 37degC for 15 - 30 minutes.
  2. Centrifuge at 2500 RPM in the Beckman CS-6R table top microtiter plate centrifuge for 5 minutes and decant supernatant.
  3. Resuspend the cells in 1.0 ml YENB or 2xTY.
  4. Add 130 ul of 20 mg/ml IPTG (in water) and 130 ul of 24 mg/ml X-Gal (in DMF).
  5. Spread one-half of the cell suspension over each of two pre-warmed "lasagna" dishes using sterile inoculating loop. The "lasagna" dishes are NUNC Bio-Assay dishes #240853 243x243x18 mm (VWR# 25384-002) that are prepared by mixing 10g Bacto-Trypotone (Difco # 0123-01-1), 5 g Bacto-Yeast extract (Difco # 0127-05-03), 10 g NaCl, and 18 g of Bacto-agar (Difco #0140-01) and brought to 1 liter with ddwater. This mixture is sterilized and cooled to 55 deg C. Then, add 10 ml of Ampicillin (10 mg/ml Sigma #A-9518 in sterile ddwater) and pour 310 ml of this LB+Amp media onto each "lasagna" plate in the sterile hood. Allow the plates to cool to room temperature before storing in the cold room.
  6. Allow 10-20 minutes for the cell suspension to diffuse into the agar, and then, invert and incubate for at least 20 hours at 37degC.
  7. Subsequently, incubate the plates in the cold room (4 degC.) for an additional 3-4 hours to intensify the blue color.
  8. Pick the colonies into 384-well flat bottom microtiter plate (NUNC #242757) containing 70 ul TB + salt supplemented with 100 ug/ml ampicilin using the Q-Pix colony picker. Also pick a few dozen colonies into an extra 96 well plate containing 200 ul media for use below to replace the contents of wells with no cell growth.
  9. Incubate the plates in a HiGro incubator (Gene Machines, Inc.) for 22 hours at 37degC with shaking at 520 rpm. The oxygenated flow is set to begin 3.5 hours after shaking begins and a full open flow rate with the HiGro Oxygen Flow Setting at 0.5 second on and 0.5 minutes off.
  10. Examine the wells and replace the contents of wells with no cell growth with culture from positive growth cells obtained from the extra plate containing additional colonies picked into a partial plate.
  11. Centrifuge the 384 well plates at 3000 RPM in the Beckman CS-6R table top microtiter plate centrifuge for 10 minutes.
  12. Decant the supernatant by inverting the plates onto 3-4 layers of paper towels and gently tappingthe inverted bottom of the plate.
  13. Freeze the plates for at least 2-3 hours or to overnight at -20degC or -80degC.

The first stage of the DNA isolation protocol (steps 14 through 21) are done using the mini-Stacatto. This machine contains two units. The Twister II robot which contains a robotic arm that moves microtiter plates from and to the storage racks mounted on the Twister II. The second unit is the Sciclone which is a liquid handling station with 384-channel pipettor head and four magnetic shakers.

  1. Remove the plates from the freezer and place them in a Zymark rack. The TwisterII robotic arm picks four plates at a time and places them on four magnetic shakers inside the Sciclone deck.
  2. A 384-channel pipettor head adds 23 ul of TE-RNase (50 mM Tris-HCl, pH 7.6, 0.5 M EDTA-Na, 40 ug/ul RNase A, T1 RNase 0.04 U/ul) solution.
  3. The megnetic shakers then shake the plates for 10 minutes at a setting of 1 (~1000 RPM).
  4. The cannula adds 23 ul of lysis buffer (1% SDS, 0.2 M NaOH). The magnetic shakers then shake the plates for additional 10 minutes at a setting of 1.
  5. Finally, the 384 head adds 23 ul of 3 M NaOAc (408.24 g NaOAc-3H2O in a total volume of 1 L adjusted to pH 4.5 with acetic acid) or 3 M K-OAc (294.45 g KOAc in a total volume of 1 L adjusted to pH 4.5 with acetic acid). The shakers shake the plates for 10 minutes at a setting of 1.
  6. Once the samples are processed, the twister arm then moves the plates from the Sciclone deck and places the in a storage rack.
  7. Place the rack in -80 deg.C freezer overnight.
  8. Thaw the plates for ~30 minutes and centrifuge at 3000 rpm for 45 minutes at 4degC in the Beckman CS-6R table top microtiter plate centrifuge.

Steps 22 through 27 are performed using the VPrep which also is a liquid handling station with 384-channel pipettor head and four movable shelves on either side of the pipettor head

  1. Using the VPrep, transfer 50 ul of the supernatant into a new 384-well plate .
  2. Add 50 ul 100% isopropanol using the VPrep. The VPrep then itroduces 50 ul air bubles to mix the isopropanol thoroughly.
  3. Centrifuge at 3000 rpm for 30 minutes at 4 deg. C in the Beckman CS-6R table top microtiter plate centrifuge.
  4. Decant supernatant by inverting the plates onto 3-4 layers of paper towels and gently tappingthe inverted bottom of the plate.
  5. Wash by adding 50ul of 70% ethanol using the VPrep and centrifuge at 3000 rpm for 10 minutes at 4 deg. C in the Beckman CS-6R table top microtiter plate centrifuge.
  6. Decant the final ethanol wash, dry the pelleted DNA for 10 minutes in a vacuum and then dissolve the final, dryed DNA in 20 ul of water (added using the VPrep) and shake on a bench-top shaker for 10 mins at a setting of 8.

Alternatively, steps 22 through 27 can be performed on the Zymark mini-staccato with minimal human intervention.

  1. The Nunc 384 plates are stacked in such a way that the each the new plate is followed by the old plate.
  2. The Sciclone 384 canula then transfers 50 ul of the supernatant to the new plate (25 ul twice).
  3. Next, the Sciclone 384 canula adds 50 ul isopropanol and mixes the contents by introducing a total of 500 ul air bubbles.
  4. Centrifuge at 3000 rpm for 30 minutes at 4 deg. C in the Beckman CS-6R table top microtiter plate centrifuge.
  5. Decant supernatant manually by inverting the plates onto 3-4 layers of paper towels and gently tapping the inverted bottom of the plate.
  6. Place the plates back into the Zymark rack to run the ethanol wash program..
  7. The Sciclone head with add 50 ul of 70% ethanol (25 ul twice) after the TwisterII arm places them on the deck.
  8. Centrifuge at 3000 rpm for 10 minutes at 4 deg. C in the Beckman CS-6R table top microtiter plate centrifuge.
  9. Decant the final ethanol wash, dry the pelleted DNA for 10 minutes in a vacuum.
  10. Place the plates back into the Zymark rack and run the water addition program that will add each plate, in sets of 4, to the SciClone shakers on the deck, add 20 ul ddwater to dissolve the final, dryed DNA using the 384 canula followed by shaking the plates for 3 minutes, and then returning them to the rack.

Note:
Typically, 4-5 ul (200 ngms) of the resulting 20 ul isolated DNA sequencing template solution are used for sequencing (with 1:20 dilution of either the ABI BigDye terminator DNA sequencing reaction mix v3.0 or the Amersham ET terminator DNA sequencing reaction mix).

Bruce Roe, broe@ou.edu