Team:Amsterdam/Notebook/Protocols/Making Competent Cells

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Contents

Preparation of chemically competent cells

Overview

We use a slightly modified version of the chemical transformation protocol used by the [http://partsregistry.org/Help:Protocols/Competent_Cells Registry of Standard Biological Parts].
This protocol is a variant of the [http://www.ncbi.nlm.nih.gov/pubmed/1943786?dopt=Abstract Hanahan protocol] using CCMB80 buffer for DH10B, TOP10 and MachI strains. It builds on Example 2 of the [http://openwetware.org/images/b/bd/Pat6855494.pdf Bloom05 patent] as well. This protocol has been tested on NEB10, TOP10, MachI and [http://openwetware.org/wiki/Talk:TOP10_chemically_competent_cells BL21(DE3)] cells. See [http://openwetware.org/wiki/Bacterial_Transformation OWW Bacterial Transformation page] for a more general discussion of other techniques. The [http://openwetware.org/images/c/c2/Pat6709852.pdf Bloom04] patent describes the use of essentially the same protocol for the Invitrogen Mach 1 cells. The [http://openwetware.org/images/0/0c/Pat6960464.pdf Jesse '464 patent] describes using this buffer for DH5α cells. Unfortunately, we have found the competence of DH5α to be far lower when compared to TOP10.

Materials

  • Detergent-free, sterile glassware and plasticware. We use dedicated centrifuge tubes that are only used for preparation of competent cells
  • Table-top OD600nm spectrophotometer
  • SOB
  • CCMB80 buffer (see below)

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 4°C
  • slight dark precipitate appears not to affect its function

Procedure

Prechill plasticware and glassware

Prechill 250mL centrifuge tubes and screw cap tubes before use.

Preparing seed stocks

  • Prepare TOP10 preculture by scraping a -80d°C TOP10 glycerol stock into 3ml of LB medium and shake overnight at 37°C

Preparing competent cells

  • Prepare TOP10 preculture by inoculating 3ml of LB medium with a -80d°C TOP10 glycerol stock into and shake overnight at 37°C
  • Inoculate 250 ml of SOB medium with 3 ml of preculture and grow in a 36°C shaker to an OD600nm of 0.3
    • This takes approximately 2.5 hours.
    • Aim for an OD600 of slightly below 0.3
  • Centrifuge at 3000g at 4°C 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
    • This is is often less than completely gentle. It still works.
  • Incubate on ice 20 minutes
  • Centrifuge again at 4°C and resuspend in 10 ml of ice cold CCMB80 buffer.
  • Test OD of a mixture of 200 μl SOC and 50 μl of the resuspended cells.
  • Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test. Usually this additional step is not necessary.
  • Incubate on ice for 20 minutes
  • Aliquot 50 μl samples to sterile, prechilled, 1.5 ml or 2 ml eppendorf tubes and flash freeze by immersion in liquid nitrogen.
  • Store at -80°C indefinitely.
  • Test competence (see below)

Measurement of competence

  • Transform 50 μl of cells with 10 pg of standard pUC19
    • This is 1 μl of standard pUC19 Invitrogen plasmid
  • Hold on ice for 30 minutes
  • Heat shock 60 sec at 42°C
  • Add 200 μl SOC
  • shake at 37°C for 2 hours in 14 ml aerated tubes
  • Plate 20 μl and 200 on AMP plates using a sterile plate spreader
    • Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA
    • We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA

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 (12.8ml/liter)
    • Previous protocol indicated amount of acetic acid added should be 23 ml/liter but that amount was found to be 2X too much per tests on 1.23.07 --Meagan 15:50, 25 January 2007 (EST)
  • 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
  • Reshma P. Shetty 10:49, 11 February 2008 (CST): Use of the ligation adjustment buffer is optional.

References

  1. Hanahan91 pmid=1943786
  2. Reusch86 pmid=3536850
  3. Addison04 pmid=15470891
  4. Bloom04 US Patent 6,709,852 [http://openwetware.org/images/c/c2/Pat6709852.pdf pat6709852.pdf]
  5. Bloom05 US Patent 6,855,494 [http://openwetware.org/images/b/bd/Pat6855494.pdf pat6855494.pdf]
  6. Jesse05 US Patent 6,960,464 [http://openwetware.org/images/0/0c/Pat6960464.pdf pat6960464.pdf]

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