(i) Competent Cell Preparation Protocol
(ii) Salmonella transformation protocol
(i) Competent Cell Preparation Protocol for E. coli, adapted from protocol 24: The Inouye Method for Preparation and Transformation of Competent E. coli in Protocols in Molecular Cloning
Author: B Kulasekara
Note: Organic contaminants can reduce the efficiency of transformation, so prerinse all glassware used in this procedure with ddH2O.
1. Grow your E. coli strain on an LB plate overnight at 37C.
2. Take a few colonies and make a thick suspension in LB. Use this to inoculate 3 1L flasks containing 250 ml LB to an OD of .001, .005, and .010 with Mach1 cells or .005, .010, and .020 with more slowly growing strains of E. coli.
3. Incubate cultures at 18 C with shaking at 225 rpm and grow for ~14 hrs (overnight).
4. After the 14 hr time period, select the culture with an OD600 within the range of 0.4 to .55, or wait until one of the cultures reaches this OD range.
5. Place culture flask in an ice water bath and incubate for 10 minutes.
6. Pour culture into large (500ml) centrifuge bottles and pellet by centrifuging at 4C at 2500g for 10 minutes.
7. Pour off medium and resuspend in chilled 35 ml Inoue Transformation Buffer and transfer to a 50 ml conical tube.
8. Pellet cells by centrifuging at 3000 g at 4C in a large table top centrifuge.
9. Repeat steps 7 and 8 once.
10. Resuspend the cells in 5ml chilled Inoue Transformation Buffer and keep on ice.
11. Add 0.375 ml DMSO dropwise to the bacterial suspension, mixing between each drop by swirling.
12. Make aliquots of a volume of your choosing and freeze in liquid nitrogen.
13. Store at -80C.
Inoue transformation buffer (chilled to 0-4 degrees)
a. Prepare 0.5M PIPES pH6.7 by dissolving 15.1 g of PIPES in 80 ml ddH2OH to 6.7 with 5M KOH. Adjust the volume to 100ml with ddH2O and sterilize by filtration.
b. Prepare the buffer by dissolving all of the solutes listed below in 800 ml of ddH2O and then add 20 ml of 0.5 M PIPES ph6.7. Adjust the volume of the Inoue transformation buffer to 1 liter with dd H2O.
|Amount per liter||Final Concentration|
|MnCl2 4H2O||10.88 g||55mM|
|CaCl2 2H2O||2.20 g||15 mM|
|KCl||18.65 g||150 mM|
|PIPES(0.5M,pH6.7)||20 ml||10 mM|
|Sterilize by filtration|
Bridget RK. Tuesday, January 24, 2012
(ii) Salmonella transformation with a plasmid DNA
Grow overnight of Salmonella
Back-dilute 1:50 (60 uL into 3 mL) LB
Incubate 37C with shaking
Pellet at high speed in epp centrifuge 30 sec
Wash in ice cold water
Repeat pellet-wash two more times (total of three water washes)
Pellet, resuspend in 40 uL
Add 0.5-1.5 uL plasmid
Recover in SOC 37C 1 hour
Plate on selective media
Ingrid SP. January 24, 2012
(iii) Growth curves, growth rate calculations. etc. Follow the hyperlink to access this protocol.