Team:Amsterdam/Notebook/Protocols/Making Competent Cells
Preparation of chemically competent cells
We use a slightly modified version of the chemical transformation protocol used by the Registry of Standard Biological Parts.
This protocol is a variant of the Hanahan protocol 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 NEB10, TOP10, MachI and BL21(DE3) cells. See OWW Bacterial Transformation page for a more general discussion of other techniques. The Bloom04 patent describes the use of essentially the same protocol for the Invitrogen Mach 1 cells. The 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.
- Prechilled, detergent-free, sterile glassware and plasticware. We use dedicated centrifuge tubes that are only used for preparation of competent cells
- Table-top OD600nm spectrophotometer
- CCMB80 buffer (see below)
- 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
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 -80°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 37°C shaker to an OD600nm of 0.3
- This takes approximately 2.5 hours.
- Aim for an OD600 of slightly below 0.3
- Prewarmed medium can shorten preparation time by up to one hour
- 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
- Note that from this point on the cells, media, bottles and glassware will have to be kept on ice at all times!! If possible it is recommended to work in a cold room.
- 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 number of colonies / (ng of DNA x partition of total volume plated) x 1000
- Competence should be at least 5x106. The higher the better.
- freely adapted from the standard chemical transformation protocol used by the Registry of Standard Biological Parts