Feb 10, 2023
Preparation and transformation of chemically super-competent Escherichia coli
This protocol is a draft, published without a DOI.
- 1University of Oslo
Protocol Citation: Andreas Sagen 2023. Preparation and transformation of chemically super-competent Escherichia coli. protocols.io https://protocols.io/view/preparation-and-transformation-of-chemically-super-cn5uvg6w
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: February 08, 2023
Last Modified: February 10, 2023
Protocol Integer ID: 76692
Keywords: Transformation, Inoue method, E. coli
Abstract
Based on a method produced by Inoue, et al.
Inoue, Nojima, H., & Okayama, H. (1990). High efficiency transformation of Escherichia coli with plasmids.Gene,96(1), 23–28. https://doi.org/10.1016/0378-1119(90)90336-P
Materials
LAF
Scale
Ice
Centrifuge
Incubator
Protocol materials
Potassium hydroxide solutionSupelcoCatalog #P4494
Step 4
Manganese(II) chloride tetrahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3634
Step 2
Potassium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P4504
Step 2
PIPESMerck MilliporeSigma (Sigma-Aldrich)Catalog #P1851
Step 2
Calcium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #C3881
Step 2
Preparation of Inoue transformation buffer
Preparation of Inoue transformation buffer
In a sterile flask, add 200 mL distilled water
Measure 2.5 mL (1 Molarity (M) ) PIPES, 2.177 g Manganese(II) chloride tetrahydrate, 3.75 mL (1 Molarity (M) ) Calcium chloride and 4.660 g Potassium chloride.
Materials:
PIPESSigma AldrichCatalog #P1851
Manganese(II) chloride tetrahydrateSigma AldrichCatalog #M3634
Calcium chlorideSigma AldrichCatalog #C3881
Potassium chlorideSigma AldrichCatalog #P4504
Add measured reagents and mix for 00:05:00
5m
Adjust pH to 6.7 with Potassium hydroxide solution
Materials:
Potassium hydroxide solutionSigma AldrichCatalog #P4494
Fill flask with distilled water to 250 mL
Filter sterilize solution with a filter (0.2 µm) and store refrigerated (4 °C )
Preparation of chemically super-competent cells
Preparation of chemically super-competent cells
Prepare a culture of E. coli on an LB agar plate. Pick a single colony, and inoculate in 500 mL S. O. C. broth in a 1000 mL flask.
Incubate at 18 °C with shaking at 100 rpm overnight, until OD600 reaches 0.6
Aliquot entire culture volume into 50 mL canonical tubes
Place tubes on ice for 00:10:00
10m
Centrifuge tubes with 5000 rcf, 4°C, 00:10:00
10m
Discard supernatant and resuspend with 16 mL CRM transformation buffer
Incubate cells on ice for 00:10:00
10m
Centrifuge tubes with 5000 rcf, 4°C, 00:10:00
10m
Discard supernatant and resuspend with 8 mL Inoue transformation buffer. Pool into two tubes
Centrifuge tubes with 5000 rcf, 4°C, 00:10:00 . Meanwhile, prepare 50 mL DMSO-Inoue transformation buffer by diluting 3.5 mL DMSO in 46.5 mL Inoue transformation buffer
Materials:
DMSOSigma AldrichCatalog #196055
10m
Discard supernatant and resuspend with 10 mL DMSO-Inoue per tube
Incubate cells on ice for 00:30:00
30m
Aliquot 100 µL cell suspension into sterile 500 µL screw cap reaction tubes (Sarstedt #72.704.200).
Note
While creating aliquots, keep original tubes, and aliquots on ice, until snap-freeze take place
Snap freeze tubes in liquid nitrogen using a floating foam tube rack (Southern labware #HS2166)
Transfer aliquots storage box, and place in an ultra-low temperature freezer or vapor-phase nitrogen tank
Note
Store tubes in 50 mL canonical tubes, or similar containers
Transformation
Transformation
1h 32m 30s
1h 32m 30s
Quickly thaw a single reaction tube with 100 µL hyper-competent cells in Inoue-DMSO
Mix 1-5 µL plasmid (ligation product)
Note
Do not exceed 5% of the volume competent cells
Note
Use up-to 25 ng per 50 µL of competent cells
Incubate cells on ice for 00:30:00
30m
Heat-shock cells at 42 °C for 00:00:30 , followed by 00:02:00 at 4 °C immediatly
2m 30s
Add 500 µL prewarmed S. O. C. medium and incubate for 37 °C at 200 rpm for 01:00:00
1h
Add desired amount of suspension on LB plates with ampicillin (100 µg/mL) and incubate overnight