Team:Amsterdam/Notebook/Protocols/Making Competent Cells

From 2011.igem.org

Preparation of chemically competent cells

Overview

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.

Materials

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
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 -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.

References

freely adapted from the standard chemical transformation protocol used by the Registry of Standard Biological Parts