Feb 11, 2023
Preparation and transformation of chemically hyper-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 hyper-competent Escherichia coli. protocols.io https://dx.doi.org/
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 04, 2023
Last Modified: February 11, 2023
Protocol Integer ID: 76363
Keywords: transformation, e. coli, Modified Inoue, preparing high efficiency transformation escherichia, high efficiency transformation escherichia, plasmid, larger dna fragment, dna
Abstract
Based on a protocol produced by Liu, et al.
Liu, Chang, W., Pan, L., Liu, X., Su, L., Zhang, W., Li, Q., & Zheng, Y. (2018). An Improved Method of Preparing High Efficiency Transformation Escherichia coli with Both Plasmids and Larger DNA Fragments.Indian Journal of Microbiology,58(4), 448–456. https://doi.org/10.1007/s12088-018-0743-z
Materials
LAF
Scale
Incubator
Heat cycler / Heat-bath
Ice
Protocol materials
Lactoferrin BMerck MilliporeSigma (Sigma-Aldrich)Catalog #L9507
Manganese(II) chloride tetrahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3634
Calcium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #C3881
PIPESMerck MilliporeSigma (Sigma-Aldrich)Catalog #P1851
Potassium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P4504
Potassium hydroxide solutionSupelcoCatalog #P4494
Dimethyl sulfoxide (DMSO)Catalog #196055
Preparation of CRM transformation buffer
In a sterile flask, add 200 mL distilled water
Measure 2.5 mL (1 Mass Percent ) PIPES, 2.177 g Manganese(II) chloride tetrahydrate, 7.5 mL (1 Mass Percent ) Calcium chloride, 4.660 g Potassium chloride and 110 µL (1 µg/mL) Lactoferrin B.
Materials:
PIPESMerck MilliporeSigma (Sigma-Aldrich)Catalog #P1851
Manganese(II) chloride tetrahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3634 c
Calcium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #C3881
Potassium chlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #P4504
Lactoferrin BMerck MilliporeSigma (Sigma-Aldrich)Catalog #L9507
Add measured reagents and mix for 00:05:00
5m
Adjust pH to 6.7 with Potassium hydroxide solution
Materials:
Potassium hydroxide solutionSupelcoCatalog #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 hyper-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 CRM transformation buffer. Pool into two tubes
Centrifuge tubes with 5000 rcf, 4°C, 00:10:00 . Meanwhile, prepare 50 mL DMSO-CRM transformation buffer by diluting 3.5 mL DMSO in 46.5 mL CRM transformation buffer
Materials:
Dimethyl sulfoxide (DMSO)Catalog #196055
10m
Discard supernatant and resuspend with 10 mL DMSO-CRM 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
1h 32m 30s
Quickly thaw a single reaction tube with 100 µL hyper-competent cells in CRM-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