Gill:Chung cells: Difference between revisions
(New page: ==Materials== *Overnight ''E. coli'' culture *50 mL Falcon tubes *LB *TSS *1.5 mL centrifuge tubes *Freezer box ==Procedure== Dilute an overnight culture of ''E. coli'' 1:100 in LB m...) |
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*LB | *LB | ||
*[[TSS]] | *[[TSS]] | ||
*1.5 mL centrifuge tubes | *1.5 mL centrifuge tubes<br> | ||
*Freezer box | Storage:<br> | ||
*Freezer box <br> | |||
Transformation:<br> | |||
*Polypropylene tubes | |||
*LB or TSS with 20 mM glucose | |||
*Plasmid DNA | |||
==Procedure== | ==Procedure== | ||
Dilute an overnight culture of ''E. coli'' 1:100 in LB media. Grow to an OD of 0.3 and then put the culture on ice. Transfer the culture to chilled 50 mL Falcon tubes and centrifuge at 5,000 g for 10 minutes at 5°C. Discard the supernatant and resuspend at 1/10th volume of cold TSS by gently pipetting up and down. Transfer 100 μL aliquots to chilled 1.5 mL centrifuge tubes, freeze in liquid nitrogen | Dilute an overnight culture of ''E. coli'' 1:100 in LB media. Grow to an OD of 0.3 and then put the culture on ice. Transfer the culture to chilled 50 mL Falcon tubes and centrifuge at 5,000 g for 10 minutes at 5°C. Discard the supernatant and resuspend at 1/10th volume of cold TSS by gently pipetting up and down. Transfer 100 μL aliquots to chilled 1.5 mL centrifuge tubes, and either use immediately or freeze in liquid nitrogen and store in the -80 freezer. | ||
To tranform the cells, add 100 uL of E. coli to a cold polypropylene tube which is kept on ice. Add 100 pg plasmid DNA, mix gently, and store at 5°C for 5-60 minutes. Add 900 uL of either TSS or LB with 20 mM glucose, and incubate the culture with shaking for one hour at 37°C. | |||
==Notes== | ==Notes== | ||
Revision as of 11:17, 20 August 2013
Materials
- Overnight E. coli culture
- 50 mL Falcon tubes
- LB
- TSS
- 1.5 mL centrifuge tubes
Storage:
- Freezer box
Transformation:
- Polypropylene tubes
- LB or TSS with 20 mM glucose
- Plasmid DNA
Procedure
Dilute an overnight culture of E. coli 1:100 in LB media. Grow to an OD of 0.3 and then put the culture on ice. Transfer the culture to chilled 50 mL Falcon tubes and centrifuge at 5,000 g for 10 minutes at 5°C. Discard the supernatant and resuspend at 1/10th volume of cold TSS by gently pipetting up and down. Transfer 100 μL aliquots to chilled 1.5 mL centrifuge tubes, and either use immediately or freeze in liquid nitrogen and store in the -80 freezer.
To tranform the cells, add 100 uL of E. coli to a cold polypropylene tube which is kept on ice. Add 100 pg plasmid DNA, mix gently, and store at 5°C for 5-60 minutes. Add 900 uL of either TSS or LB with 20 mM glucose, and incubate the culture with shaking for one hour at 37°C.
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- This protocol is adapted from Chung and Miller (1989). One-step preparation of competent Escherichia coli: Transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sci., 86(April), 2172–2175.
- When making TSS, first add PEG to LB, heat in a 50°C water bath, and then swirl to mix. Add Mg2+, wait until the solution gets to room temperature before adding DMSO, and filter sterilize. Store in the 5 degree fridge.
