384-well DNA Sequencing Template Picking, Growth and Isolation using the Flexys Colony Picker, HiGro Oxygenated Shaker Incubator and Hydra 96 Automated Pipettor with Moving Stage

Shaoping Lin, HongGui Jia, HongMin Wu, and B. A. Roe
Version 1 - updated 02-13-02
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 Flexys 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.
  14. Remove the plates from the freezer and using the Hydra, add 25 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 and shake on a bench-top shaker for 30 minutes at a setting of 10.
  15. Add 25 ul of lysis buffer (1% SDS, 0.2 M NaOH) and shake on the bench-top shaker for 30 minutes at a setting of 8.
  16. Add 25 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) and shake in the HiGro at 37degC and a shaker setting of 520 rpm for 30 minutes.
  17. Freeze overnight at -80degC.
  18. 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.
  19. Transfer 40 ul of the supernatant into a new 384-well plate (NUNC #242757).
  20. Add 40 ul 100% isopropanol using the Hydra. Use the Hydra protocol with the 100 ul "air gap" to insure thorough mixing of the isopropanol and the DNA containing supernatant.
  21. Let stand at room temperature for 3-5 minutes and then centrifuge at 3000 rpm for 30 minutes at 4 deg. C in the Beckman CS-6R table top microtiter plate centrifuge.
  22. Decant supernatant by inverting the plates onto 3-4 layers of paper towels and gently tappingthe inverted bottom of the plate. Then, immediately wash by adding 50ul room temperature or 4degC 70% ethanol using the Hydra and centrifuge at 3000 rpm for 10 minutes at 4 deg. C in the Beckman CS-6R table top microtiter plate centrifuge.
  23. 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 and shake on a bench-top shaker for 30 mins at a setting of 8.
Note:
Typically, 3-4 ul (200 ngms) of the resulting 20 ul isolated DNA sequencing template solution are used for sequencing with 1:16 dilution of BigDye v3.0 mix.


Home Page

Bruce Roe, broe@ou.edu