Lactobacillus transformation (Speer 2012)

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Notes)
Current revision (15:27, 18 November 2011) (view source)
(Notes)
 
(6 intermediate revisions not shown.)
Line 3: Line 3:
Back to [[Richard Lab:protocols]]<br>
Back to [[Richard Lab:protocols]]<br>
==Overview==
==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 [[Electro-transformation of Lactobacillus spp.|Lactobacillus transformation archive]].  
+
This is basically a combination of the version of the [[Lactobacillus transformation (Berthier 1996)| Berthier (1996)]] protocol and the Mason (2005) protocol.  This protocol is designed for use with ''Lactobacillus plantarum'' and an Eppendorf 2510 electroporator.  For other protocols check out the [[Electro-transformation of Lactobacillus spp.|Lactobacillus transformation archive]].
==Materials==
==Materials==
Line 16: Line 16:
##Inoculate 5ml MRS medium with ''L. plantarum'' freezer stock.
##Inoculate 5ml MRS medium with ''L. plantarum'' freezer stock.
##Grow overnight at 30°C without shaking.
##Grow overnight at 30°C without shaking.
-
#'''Make 100ml 2% glycine:'''
+
#'''Make 100ml 2.5% glycine:'''
-
##Add 1g glycine to two flasks of 50ml MRS medium.
+
##Add 1.25g glycine to two flasks of 50ml MRS medium.
##Shake to dissolve.
##Shake to dissolve.
#'''Grow cells:'''
#'''Grow cells:'''
Line 28: Line 28:
#'''Treat with ice-cold EDTA:'''
#'''Treat with ice-cold EDTA:'''
##Centrifuge for 5min at 4000g
##Centrifuge for 5min at 4000g
-
##Resuspend in 10ml 50mM EDTA.
+
##Resuspend in 5ml 50mM EDTA.
##Incubate on ice for 5 minutes
##Incubate on ice for 5 minutes
 +
##Add 25ml ice-cold DI water.
#'''Wash with ice-cold DI water:'''
#'''Wash with ice-cold DI water:'''
##Centrifuge culture for 5min at 4000g
##Centrifuge culture for 5min at 4000g
Line 61: Line 62:
==Notes==
==Notes==
*All of the liquid reagents/buffers should be autolaved before use.
*All of the liquid reagents/buffers should be autolaved before use.
 +
*Work fast and keep the tubes on ice if you're going to pause for any reason.
*This protocol works well for chromosomal insertion
*This protocol works well for chromosomal insertion
*Notice the high amount of DNA added to the competent cells (10%)
*Notice the high amount of DNA added to the competent cells (10%)
Line 70: Line 72:
==Contact==
==Contact==
-
*mas853@psu.edu
+
*Tom Richard (tlr20@psu.edu)
 +
*Mike Speer (mas853@psu.edu)
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  
or instead, [[Talk:{{PAGENAME}}|discuss this protocol]].  

Current revision

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

Contents

Overview

This is basically a combination of the version of the Berthier (1996) protocol and the Mason (2005) protocol. This protocol is designed 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 2.5% glycine:
    1. Add 1.25g 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 5ml 50mM EDTA.
    3. Incubate on ice for 5 minutes
    4. Add 25ml ice-cold DI water.
  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.
  • Work fast and keep the tubes on ice if you're going to pause for any reason.
  • 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. -->

Personal tools