Gill:Chung cells

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(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...)
Current revision (16:51, 7 November 2013) (view source)
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*LB
*LB
*[[TSS]]
*[[TSS]]
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*1.5 mL centrifuge tubes
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*1.5 mL centrifuge tubes<br>
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*Freezer box
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Storage:<br>
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*Freezer box <br>
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Transformation:<br>
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*LB or TSS with 20 mM glucose
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*Plasmid DNA
==Procedure==
==Procedure==
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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, and then store in the -80 freezer.
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#Dilute an overnight culture of ''E. coli'' 1:100 in LB media. Grow to an OD of 0.3 and 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 with 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 1-3 μL prepped plasmid DNA to a 100 μL aliquot of Chung cells (first thaw on ice if frozen), mix gently, and store at 5°C for 5-60 minutes. Don't forget to set up a control using sterile water instead of plasmid DNA.
 +
#Add 900 μL of either TSS or LB with 20 mM glucose, and incubate the culture with shaking for one hour at 37°C (larger vectors may require a longer recovery period).
 +
#Plate 10 μL, 100 μL, and the rest of the transformation on selective media.
==Notes==
==Notes==

Current revision

Materials

  • Overnight E. coli culture
  • 50 mL Falcon tubes
  • LB
  • TSS
  • 1.5 mL centrifuge tubes

Storage:

  • Freezer box

Transformation:

  • LB or TSS with 20 mM glucose
  • Plasmid DNA

Procedure

  1. Dilute an overnight culture of E. coli 1:100 in LB media. Grow to an OD of 0.3 and put the culture on ice.
  2. Transfer the culture to chilled 50 mL Falcon tubes and centrifuge at 5,000 g for 10 minutes at 5°C.
  3. Discard the supernatant and resuspend at 1/10th volume with cold TSS by gently pipetting up and down.
  4. 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.
  5. To tranform the cells, add 1-3 μL prepped plasmid DNA to a 100 μL aliquot of Chung cells (first thaw on ice if frozen), mix gently, and store at 5°C for 5-60 minutes. Don't forget to set up a control using sterile water instead of plasmid DNA.
  6. Add 900 μL of either TSS or LB with 20 mM glucose, and incubate the culture with shaking for one hour at 37°C (larger vectors may require a longer recovery period).
  7. Plate 10 μL, 100 μL, and the rest of the transformation on selective media.

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.
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