IGEM:The Citadel/Notebook/Notes Brian/05-29-10

From OpenWetWare

Jump to: navigation, search

Contents

May 29

Today we worked towards making our own electrocompetent cells.

  • Observed Adam's Miniprep Technique as a refresher for later this week.
  • Prepared 10% glycerol.
  • Prepared 600ml ddH2O.
  • Observed plate and slant of M.luteus made on 05-28-10.
  • Finished taking inventory.
    • Talked with Dr. Donnell about things we would need for TK's Electrocompetency Protocol.
  • Calculated RPMs for our centrifuge for the Knight Lab protocol.
  • Prepared for Electrocompetency Protocol execution in two days: 05-31-10. The protocol is below.
  • Based on the warning from Reshma in the protocol, we wanted to test our flat-bottomed 250 mL tubes. These were the closest item we had to what the protocol called for, so we needed to ensure that they would hold up under the required stress. The instructional insert for our tube claimed that they could withstand up to 4000 RCF at 4°C, but to be sure...
    • We've tested Nalgene®'s 250 mL flatbottomed tubes (catalog #3127-0250) in Fischer Scientific's Marathon 21000R Centifuge at 2000 RCF while cooling to 4°C. We experienced no broken tubes in our tests and will update here if one does break.


Protocol for Electrocompetent Cells

Adapted from the Knight Lab protocol by Austin Che with an annotation by Reshma Shetty.

Materials

  • -80°C freezer
  • 37°C incubator
  • Refrigerated centrifuge that accepts 225 mL culture tubes (We'll use 250mL)
  • ~500 mL LB Lennox supplemented with antibiotic if appropriate (We'll use LB Miller out of necessity)
  • ~600 mL sterile deionized water chilled to 4°C
  • 50 mL sterile 10% glycerol in deionized water chilled to 4°C
  • Ice bucket and ice
  • Dry ice, ethanol bath
  • Many 1.5 mL plastic tubes chilled to -80 °C
  • 14 mL culture tube for starter culture
  • 2 L flask for culture (We'll use two 1 L flasks)
  • 225 mL plastic tubes for centrifugation (we'll use 250mL)
  • Pipets

Procedure

  1. Prechill all tubes and pipets at 4°C or -80°C as appropriate.
    Also rinse all flasks with H2O prior to autoclaving in order to remove residual detergents that may remain on glassware from dishwashing. This step may increase competency. Autoclaving with water, which is then discarded, is even better.
  2. Inoculate 5mL LB medium and grow overnight at 37°C with rotation.
    Use LB Lennox rather than LB Miller in order to lessen salt content of media
  3. Add the 5mL overnight culture to 450mL LB medium and incubate at 37°C with vigorous shaking until the OD 600nm is between 0.5 and 1.0. It should take about 3 hours.
    For recA- strains, the OD 600 nm should be between 0.5 and 0.7 according to one online source.
  4. Fast cool the centrifuge with the correct rotor to 4°C
  5. Pour the culture into two 225 mL centrifuge tubes.
  6. Place the tubes on ice for 15 minutes.
    This step can vary in incubation time between 15 minutes and 1 hr. Longer incubation times may lead to higher competency.
    For the following steps it is important to keep cells cold and remove all the supernatant in each step to remove residual ions.
  7. Centrifuge for 10 mins at 2000g at 4°C (That's 3282.4 RPMs on our machine.)
  8. Remove supernatant and gently resuspend pellets with 200mL cold sterile water.
    Initially add 10-20 mL of water and resuspend by pipetting. Then add the rest of the water.
  9. Centrifuge for 15 mins at 2000g at 4°C (That's 3282.4 RPMs on our machine.)
  10. Remove supernatant and gently resuspend pellets with 200mL cold sterile water.
    Initially add 10-20 mL of water and resuspend by pipetting. Then add the rest of the water.
  11. Hold on ice for 30 minutes
  12. Centrifuge for 15 mins at 2000g at 4°C (That's 3282.4 RPMs on our machine.)
  13. Remove supernatant and gently resuspend pellets with 25mL cold 10% glycerol.
    This can be optionally transferred to a 50 mL conical tube.
  14. Hold on ice for 30 minutes
  15. Centrifuge for 15 mins at 1500g at 4°C (That's 2844 RPMs on our machine.)
  16. Remove the supernatant and add 500 μl of 10% glycerol
  17. Resuspend the cells in a final volume of approximately 1 ml
  18. Aliquot 50 μL per tube (tubes on ice)
  19. Shock freeze cell suspensions in a dry ice and ethanol bath.
    One website recommended against using liquid nitrogen but did not justify this recommendation.
  20. Store at -80°C

NOTE: Use the conical-bottomed tubes over the flat-bottomed ones for the swinging buckets in the centrifuge to prevent the tubes from breaking.

Personal tools