TOP10 chemically competent cells

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(5x Ligation Adjustment Buffer)
Line 74: Line 74:
* Glycerol 46.8%  (468 ml/liter)
* Glycerol 46.8%  (468 ml/liter)
* pH adjustment with 2.3%  of a 10% acetic acid solution (23 ml/liter)
* pH adjustment with 2.3%  of a 10% acetic acid solution (23 ml/liter)
 +
* water to 1 liter
 +
* autoclave or sterile filter
* Test pH adjustment by mixing 4 parts ligation buffer + 1 part 5x ligation adjustment buffer and checking pH to be 6.3 - 6.5
* Test pH adjustment by mixing 4 parts ligation buffer + 1 part 5x ligation adjustment buffer and checking pH to be 6.3 - 6.5
-
 
==References==
==References==

Revision as of 14:16, 27 November 2006

This protocol is a variant of the Hanahan protocol [1] using CCMB80 buffer for DH10B, TOP10 and MachI strains. It builds on Example 2 of the Bloom05 patent as well. This protocol has been tested on TOP10 and MachI cells. See Bacterial Transformation for a more general discussion of other techniques. The Jesse '464 patent describes using this buffer for DH5α cells. The Bloom04 patent describes the use of essentially the same protocol for the Invitrogen Mach 1 cells.

Contents

Preparing glassware and media

Detergent is a major inhibitor of competent cell growth and transformation. Glass and plastic must be detergent free for these protocols. The easiest way to do this is to avoid washing glassware, and simply rinse it out. Autoclaving glassware filled 3/4 with DI water is an effective way to remove most detergent residue. Media and buffers should be prepared in detergent free glassware and cultures grown up in detergent free glassware.

Preparing seed stocks

  • streak TOP10 cells on an SOB plate and grow for single colonies at 23 C
    • room temperature works well
  • Pick single colonies into 2 ml of SOB medium and shake overnight at 23 C
    • room temperature works well
  • Add glycerol to 15%
  • Aliquot 1 ml samples to Nunc cryotubes
  • Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 5 minutes
    • This step may not be necessary
  • Place in -80 freezer indefinitely.

Preparing competent cells

  • Inoculate 250 ml of SOB medium with 1 ml vial of seed stock and grow at 20 C to an OD of 0.3
    • This takes approximately 16 hours.
    • Controlling the temperature makes this a more reproducible process, but is not essential.
    • Room temperature will work. You can adjust this temperature somewhat to fit your schedule
    • Aim for lower, not higher OD if you can't hit this mark
  • Centrifuge at 3000g / 4C for 10 minutes in a flat bottom centrifuge bottle.
    • Flat bottom centrifuge tubes make the fragile cells much easier to resuspend
    • It is often easier to resuspend pellets by mixing before adding large amounts of buffer
  • Gently resuspend in 80 ml of ice cold CCMB80 buffer
    • sometimes this is less than completely gentle. It still works.
  • Incubate on ice 20 minutes
  • Centrifuge again at 4C and resuspend in 20 ml of ice cold CCMB80 buffer.
  • Incubate on ice for 20 minutes
  • Aliquot to chilled screw top 2 ml vials or 50 μl into chilled microtiter plates
  • Store at -80C indefinitely.
    • Flash freezing does not appear to be necessary
  • Test competence (see below)
    • Good cells should yield around 100 - 400 colonies

CCMB80 buffer

  • 10 mM KOAc pH 7.0 (10 ml of a 1M stock/l)
  • 80 mM CaCl2.2H2O (11.8 g/l)
  • 20 mM MnCl2.4H2O (4.0 g/l)
  • 10 mM MgCl2.6H2O (2.0 g/l)
  • 10% glycerol (100 ml/l)
  • adjust pH DOWN to 6.4 with 0.1N HCl if necessary
    • adjusting pH up will precipitate manganese dioxide from Mn containing solutions.
  • sterile filter and store at 4C
  • slight dark precipitate appears not to affect its function

Measurement of competence

  • Transform 50 μl of cells with 1 μl of standard pUC19 plasmid (Invitrogen)
    • This is at 10 pg/μl or 10-5 μg
  • Hold on ice 0.5 hours
  • Heat shock 60 sec at 42C
  • Add 250 μl SOC
  • Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated
    • using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
    • For our plasmids (pSB1AC3, pSB1AT3) which are chloramphenicol and tetracycline resistant, we find growing for 2 hours yields many more colonies
    • Ampicillin and kanamycin appear to do fine with 1 hour growth
  • Plate 20 μl on AMP plates using 3.5 mm glass beads
  • Transformation efficiency is 15 x colony count x 105
  • There is anecdotal evidence that refreezing partially used cell aliquots dramatically reduces transformation efficiency.


5x Ligation Adjustment Buffer

  • Intended to be mixed with ligation reactions to adjust buffer composition to be near the CCMB80 buffer
  • KOAc 40 mM (40 ml/liter of 1 M KOAc solution, pH 7.0)
  • CaCl2 400 mM (200 ml/l of a 2 M solution)
  • MnCl2 100 mM (100 ml/l of a 1 M solution)
  • Glycerol 46.8% (468 ml/liter)
  • pH adjustment with 2.3% of a 10% acetic acid solution (23 ml/liter)
  • water to 1 liter
  • autoclave or sterile filter
  • Test pH adjustment by mixing 4 parts ligation buffer + 1 part 5x ligation adjustment buffer and checking pH to be 6.3 - 6.5

References

  1. Hanahan D, Jessee J, and Bloom FR. . pmid:1943786. PubMed HubMed [Hanahan91]
  2. US Patent 6,709,852 Media:pat6709852.pdf [Bloom04]
  3. US Patent 6,855,494 Media:pat6855494.pdf [Bloom05]
  4. US Patent 6,960,464 Media:pat6960464.pdf [Jesse05]
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