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| Protocol for growing cells for ribosome preps 4/1/2010
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| Written by Anne Bunner, based on protocol used by Michael Jewett and Ruben Gonzalez. Edited by Michael Jewett and Brian Fritz.
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| Day 0 – Streak fresh plate of MRE600 cells Time required: 0.5 hours
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| Day 1 – Making media and inoculating starters Time required: 4 hours
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| 2XYT for 1 L of media (for 4L) (for 250 mL: 2 starters and blank)
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| 16g tryptone 64g 4g
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| 10g Yeast extract 40g 2.5g
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| 5g NaCl 20 1.25g
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| pH to 7.2 using 1M or 5M NaOH, or use KOH if NaOH is not available.
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| If using Tunair filter flasks, put foil over the filters before autoclaving. Keep autoclaved flasks at room temperature overnight. Use 250-500 mL flasks with 100 mL culture in each for starters, and autoclave extra 50 mL in bottle or beaker for use as blank for spec.
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| Also should autoclave:
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| • 800 mL beaker for scooping ice
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| • Spatula for dispensing ice
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| • Something for measuring cells going into bottles that is safe to autoclave
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| • 100-200 mL bottle for Buffer A
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| • Centrifuge bottles (if possible)
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| Note: Polycarbonate bottles such as those for the SLC-6000 cannot be autoclaved.
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| Make Buffer A, if needed. Put bottle of Buffer A in cold room.
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| Buffer A Stock Volume per 500 mL Notes:
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| 20 mM Tris HCl, pH=7.2 @ 4°C 1 M 10 mL Needd 125 mL for
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| 100 mM NH4Cl 3 M 16.7 mL harvesting cells
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| 10 mM MgCl2 4.9 M 1020 L
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| 0.5 mM EDTA 0.5 M 500 L
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| 6 mM BME 14.3 M 210 L*
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| * add just before use
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| Inoculate 100 mL 2xYT with MRE600 cells from a freshly streaked plate. Frozen stock is ok, but plate is preferred. Shake at 37°C overnight (~16 h) at 190-225 rpm.
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| Day 2 – Growing and harvesting cells Time required: 7 hours
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| 1. Put the Tunair flasks in the shaker to pre-warm 15 min.
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| 2. Turn on spectrophotometer visible lamp. Check OD 600 nm of overnight culture. (Visible lamp does not need to warm up.)
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| 3. Before inoculation, record pH of media.
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| 4. Inoculate 4x1L with 0.2 mL overnight culture and 4x1L with 0.5 mL overnight culture each.
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| 5. Place flasks in shaker at 225 rpm at 37 C.
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| 6. Make sure Buffer A is chilling.
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| 7. Make sure rotors are chilling. Will need Sorvall SLC-6000 (large fiberglass) and eppendorf F34.
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| 8. Turn on Sorvall centrifuge and chill to 4 C.
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| 9. After 1 hour, start checking the OD 600nm of every flask regularly using media as blank. Growth usually takes 3-4 hours.
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| 10. When the OD reaches 0.1, fill sink in cold room halfway with ice. Also put 6 centrifuge bottles (autoclaved if possible) in cold room.
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| 11. When cells get to 0.5 OD (typically ~3-4 hours), transfer flask to ice and add 700-800 mL ice per liter using autoclaved beaker and spatula. Begin transferring cells to centrifuge bottles and preparing to harvest immediately after adding ice.
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| 12. For SLC-6000 rotor, use 750 mL media per bottle. Measure media with autoclaved graduated container. Balance bottles with scale near centrifuge.
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| 13. Spin cells at 6000 rpm for 10 min to harvest.
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| 14. While cells are spinning, weigh two 50 ml falcon tubes and record weights. Also, aliquot out 125 mL of cold Buffer A and add BME (52.4 uL in 125 mL).
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| 15. Discard supernatant into 4 L waste beaker.
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| 16. After cells are all harvested, add 15 mL cold Buffer A to each tube and resuspend pellets.
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| 17. Transfer cells to two pre-weighed 50 mL falcon tubes.
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| 18. Spin in eppendorf benchtop centrifuge in F34 rotor with adapters at 6100 rpm (~5000g) for 12 min to pellet. (Rotor lid will not go on but don’t worry about it. Just be sure to tighten rotor down with hex screw located over the centrifuge)
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| 19. Resuspend pellets using ~15-20 mL Buffer A each and combine into single tube. Do quantitative transfer with buffer to get all cells into single tube.
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| 20. Spin at 6100 rpm (~5000g) for 12 min to pellet. During spin, go get liquid nitrogen (located near bridge between Cook and Hogan on 2nd floor).
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| 21. Pour off supernatant and drain pellet upside down on paper towel in cold room 2-5 min.
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| 22. Weigh pellet. Base weight on original mass of weighed tube. Typical yields are 3-4 g per 4L, doubling time of ~20 minutes.
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| 23. Score pellet by dragging a small pipette tip through it as if to draw a pound sign.
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| 24. Label tube with name, date, and strain, and freeze cells in liquid nitrogen. Store at -80 C
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| 25. Don’t forget to:
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| • Turn of spectrophotometer lamp
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| • Turn off Sorvall centrifuge
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| • Clean out Sorvall rotor
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| • Turn off shaker.
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