Lactobacillus transformation (Speer 2012)

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(Contact)
(Contact)
Line 70: Line 70:
==Contact==
==Contact==
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*tlr20@psu.edu
+
*Tom Richard (tlr20@psu.edu)
-
*mas853@psu.edu
+
*Mike Speer (mas853@psu.edu)
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  

Revision as of 17:21, 16 September 2011

Back to Protocols
Back to Electro-transformation of Lactobacillus spp.
Back to Richard Lab:protocols

Contents

Overview

This is basically a modified version of the Mason (2005 protocol) for use with Lactobacillus plantarum and an Eppendorf 2510 electroporator. For other protocols check out the Lactobacillus transformation archive.

Materials

  • MRS media
  • Glycine
  • Milipore water
  • 50mM EDTA solution
  • Electroporation Buffer (0.5M Sucrose, 10% glycerol)

Procedure

  1. Grow L. plantarum
    1. Inoculate 5ml MRS medium with L. plantarum freezer stock.
    2. Grow overnight at 30°C without shaking.
  2. Make 100ml 2% glycine:
    1. Add 1g glycine to two flasks of 50ml MRS medium.
    2. Shake to dissolve.
  3. Grow cells:
    1. Add 1ml overnight culture to each flask (1:50 dlution).
    2. Culture for ~3 hours at 37°C with shaking.
  4. Wash 2x with ice-cold DI water:
    1. Centrifuge culture for 5min at 4000g
    2. Resuspend in 25ml ice-cold DI water.
    3. Repeat.
  5. Treat with ice-cold EDTA:
    1. Centrifuge for 5min at 4000g
    2. Resuspend in 10ml 50mM EDTA.
    3. Incubate on ice for 5 minutes
  6. Wash with ice-cold DI water:
    1. Centrifuge culture for 5min at 4000g
    2. Resuspend in 25ml ice-cold DI water.
  7. Wash 2x with Electroporation Buffer:
    1. Centrifuge culture for 5min at 4000g
    2. Resuspend in 25ml ice-cold 0.5M Sucrose, 10% glycerol.
    3. Repeat.
  8. Concentrate cells:
    1. Centrifuge culture for 5min at 4000g
    2. Resuspend in 0.8ml ice-cold 0.5M Sucrose, 10% glycerol.
  9. Divide cells:
    1. Aliquot 90μL of cell concentrate into ~20 microcentrifuge tubes.
    2. Keep on ice until use (within the next two hours).
  10. Add DNA:
    1. Add 10μL of plasmid DNA to the 90μL of cell concentrate.
    2. Keep on ice for 5 minutes.
    3. Put 1mm cuvettes on ice too.
  11. Electroporate:
    1. Pipette the cell/DNA mixture into the cuvette
    2. Electrporate at 1200 volts
    3. Time constant should be ~5.0
  12. Recovery:
    1. Immediately transfer the electroporated cells to 900μL of MRS medium.
    2. Incubate at 30°C for 2-3 hours.
  13. Select:
    1. Plate on medium with the appropriate antibiotic
    2. Incubate at 30°C for two days.
    3. Pick colony

Notes

  • All of the liquid reagents/buffers should be autolaved before use.
  • This protocol works well for chromosomal insertion
  • Notice the high amount of DNA added to the competent cells (10%)
  • While glycerol in the electroporation buffer is typically used to make frozen competent cells, this is not advised and will lead to decreased competency. Instead the glycerol addition is strictly to increase to competency of the cells. The reason behind this correlation is not understood.

References

There will be one here soon.


Contact

  • Tom Richard (tlr20@psu.edu)
  • Mike Speer (mas853@psu.edu)

or instead, discuss this protocol. -->

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