DNA Sequencing Template Picking, Growth and Isolation using the Flexys Colony Picker, HiGro Oxygenated Shaker Incubator and Hydra 96.
Latest update 5-23-01

After transformation, follow the steps below:
  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 0.6 ml YENB or 2xTY.
  4. Add 90 ul of 20 mg/ml IPTG (in water) and 90 ul of 24 mg/ml X-Gal (in DMF).
  5. Spread the cell suspension in 130 ul aliquots using sterile inoculating loop over four pre-warmed LB-amp agar plates poured specifically for the colony picker to contain 30 to 35 ml of LB-amp agar in a 100 x 15 mm plastic petri dish (e.g. VWR Cat. No. 25384-70).
  6. Allow 10-20 minutes for the liquid 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 96-well flat bottom microtiter plate (Dynatech Cat. No. 001-012-9200 12.7 x8.5 cm) containing 150 ul TB + salt supplemented with 100 ug/ml ampicilin using the Flexys colony picker.
  9. Incubate the plates in a HiGro(TM) incubator (Gene Machines, Inc.) for 18 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. Centrifuge the plates at 2500 ROM in the Beckman CS-6R table top microtiter plate centrifuge for 10 minutes.
  11. Decant the supernatant and freeze for at least 2-3 hours at -80degC
  12. Using the Hydra, add 60 ul of TE-RNase (50 mM Tris-HCl, pH 7.6, 0.5 M EDTA-Na, 40 mg/ml RNase A) solution and shake on a bench-top shaker for 20 minutes at a setting of 7.
  13. Add 60 ul of lysis buffer (1% SDS, 0.2 M NaOH) and shake on the bench-top shaker for 20 minutes at a setting of 5-6.
  14. Add 60 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 600 rpm for 30 minutes.
  15. Freeze overnight at -80degC.
  16. Thaw the plates for ~40 minutes and centrifuge at 3000 rpm for 45 minutes at 4degC in the Beckman CS-6R table top microtiter plate centrifuge.
  17. Transfer 60 ul of the supernatant into V-bottom microtiter plate (e.g. Dynex brand VWR Cat. No. 62402-914).
  18. Add 130 ul 95% ethanol using the 12-channel manually pipeter at room temperature.
  19. 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.
  20. Decant supernatant and wash with 200 ul 70% ethanol using the Hydra and the table-top shaker at a setting of 5-6.
  21. Centrifuge at 3000 rpm for 10 minutes at 4 deg. C in the Beckman CS-6R table top microtiter plate centrifuge.
  22. Decant the final ethanol wash, dry the pelleted DNA for 10 minutes in a 55degC oven, and then disolve the final, dryed DNA in 20 ul of water.

Note:
Typically, 4 ul (200 ngms) of the resulting 20 ul isolated DNA sequencing template solution are used for sequencing with 1:12 dilution of BigDye mix.


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Bruce Roe, broe@ou.edu