May 22, 2026
Version 2
Preparing CCMB80 Chemically Competent Cells (TOP10/DHB10) V.2
- 1University of Cambridge;
- 2Pontificia Universidad Católica
- Reclone.org (The Reagent Collaboration Network)
- Open Bioeconomy Lab
Protocol Citation: Jennifer Molloy, Jiayi Hou, Severine Cazaux 2026. Preparing CCMB80 Chemically Competent Cells (TOP10/DHB10). protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7nzqklwz/v2Version created by Jennifer Molloy
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 17, 2026
Last Modified: May 22, 2026
Protocol Integer ID: 315265
Keywords: Competent Cells, E. coli, preparing ccmb80, ccmb80 buffer for dh10b, buffer for dh5α cell, chemical transformation protocol, bacterial transformation, using ccmb80 buffer, dh5α cell, bloom05 patent, dhb10, registry of standard biological part, competent cell, dh10b, cell, same protocol for the invitrogen mach, hanahan protocol, variant of the hanahan protocol, standard biological part, bloom04
Abstract
This is the chemical transformation protocol used by Tom Knight and the Registry of Standard Biological Parts.
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, MachI and BL21(DE3) 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.
Materials
General
- Detergent-free, sterile glassware and plasticware (see procedure)
- Table-top OD600nm spectrophotometer
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
- Note: you can buy pre-made CCMB80 buffer from Teknova
Preparing Glassware and Media
Prepare detergent-free glassware
Autoclave necessary glassware filled 75% with deionised water.
Note
Detergent is a major inhibitor of competent cell growth and transformation. Media and buffers should be prepared in detergent free glassware and cultures grown up in detergent free glassware.
The easiest way to do this is to avoid washing glassware, and simply rinse it out. Autoclaving glassware filled 75% with DI water is an effective way to remove most detergent residue.
Prechill plasticware and glassware
Prechill 250 mL L250 mL centrifuge tubes and screw cap tubes before use by placing them in a -20 °C freezer or On ice .
CCMB80 Buffer Preparation
This protocol outlines the preparation of a CCMB80 buffer with specific concentrations of potassium acetate, calcium chloride, manganese chloride, magnesium chloride, and glycerol. The buffer is adjusted to a pH of 6.4 to 7.0 and is sterile filtered for storage at 4 °C .
Total duration for the entire protocol is approximately 00:50:00 .
Note
The presence of slight dark precipitate does not affect the buffer's functionality.
Safety information
- Wear appropriate personal protective equipment (PPE) including gloves, goggles, and lab coat.
- Handle all reagents with care, particularly manganese chloride and hydrochloric acid, as they are toxic and corrosive.
Expected result
The buffer should be a clear solution with no precipitate.
If precipitation occurs during pH adjustment, ensure pH is adjusted downwards only; avoiding raising pH should prevent manganese dioxide precipitation.
If the buffer does not dissolve completely, ensure adequate stirring and check the purity of reagents.
Summary of Stock Reagents
| Reagent Type | Name | Storage Conditions | Purity | Concentration | Solubility | Description | Safety Information | |
| Buffer | Potassium Acetate | Room Temperature | 99% | Prepare 1 M by dissolving 9.8 g in 100mL of dH2O, then filter-sterilize. | Soluble in water | Used to maintain pH and ionic strength | Irritant, handle with care | |
| Salt | Calcium Chloride | Room Temperature | 99% | Powder | Soluble in water | Provides calcium ions for biochemical reactions | Harmful if ingested | |
| Salt | Manganese Chloride | Room Temperature | 99% | Powder | Soluble in water | Provides manganese ions for biochemical reactions | Toxic, handle with gloves | |
| Salt | Magnesium Chloride | Room Temperature | 99% | Powder | Soluble in water | Provides magnesium ions for biochemical reactions | Irritant, handle with care | |
| Stabilizer | Glycerol | Room Temperature | 99% | 100% | Soluble in water | Acts as a cryoprotectant and stabilizer | Non-toxic, but avoid ingestion |
Equipment
- pH meter
- Magnetic stirrer
- Sterile filtration unit
- Graduated cylinder
- Weighing balance
- Beakers (200 ml)
Buffer Preparation Summary
| Item | Specifications | Quantity | |
| Potassium Acetate | 1 M stock solution | 1.0 ml | |
| Calcium Chloride | CaCl2·2H2O | 1.18 g | |
| Manganese Chloride | MnCl2·4H2O | 0.40 g | |
| Magnesium Chloride | MgCl2·6H2O | 0.20 g | |
| Glycerol | 100% glycerol | 10 ml | |
| Distilled Water | N/A | Up to 100 ml | |
| Hydrochloric Acid | 0.1 N HCl | As needed |
Buffer Preparation Protocol
- In a200 mL beaker, add 50 mL of distilled water, then 1 mL of 1 Mass Percent potassium acetate solution.
- Add 1.18 g of calcium chloride dihydrate (CaCl2·2H2O), 0.4 g of manganese chloride tetrahydrate (MnCl2·4H2O), and0.2 g of magnesium chloride hexahydrate (MgCl2·6H2O) to the beaker.
- Add 10 mL of glycerol.
- Add distilled water to bring the total volume to 100 mL
- Stir the solution using a magnetic stirrer until all solids are completely dissolved.
pH Adjustment
- Measure the pH of the solution using a pH meter.
- If the pH is above 7.0 , slowly add 0.1 Molarity (M) HCl dropwise while stirring until the pH reaches 6.4.
- If the pH is below 7.0, no adjustment is necessary.
Note
Adjusting the pH upwards will precipitate manganese dioxide; ensure to only adjust downwards if necessary.
Sterile Filtration
- Filter the prepared buffer solution through a sterile filtration unit to ensure sterility.
- Collect the filtered buffer in a sterile container.
Note
Ensure all equipment used for filtration is sterile to prevent contamination.
Quality Control
| Quality Control Measure | Criteria | |
| pH Measurement | pH must be 6.4 to 7.0 | |
| Sterility Check | No microbial growth in the buffer after incubation |
Preparing Seed TOP10/DH10B Glycerol Stocks
Streak TOP10/DH10B cells on an SOB plate and grow for single colonies at 23 °C or Room temperature
Pick single colonies into 2 mL of SOB medium and shake overnight at 23 °C or Room temperature
Add glycerol to 15 % (v/v)
Aliquot 1 mL samples to Nunc cryotubes
[Optional] Place tubes into a zip lock bag, immerse bag into a dry ice/ethanol bath for 00:05:00
Place in -80 °C freezer indefinitely.
Preparing Competent Cells
20m
Inoculate250 mL of SOB medium with1 mL vial of seed stock and grow at20 °C or Room temperature to an OD 600nm of 0.3 for approximately 16:00:00 , measuring OD with a spectrophotometer or plate reader every 30-60 min from around 12 hours until you have a good sense of how long to leave the culture.
Note
You can adjust the incubation temperature somewhat to fit your schedule. Room temperature will work; controlling the temperature makes this a more reproducible process, but is not essential.
Aim for lower, not higher OD if you can't hit the OD600nm of 0.3 mark.
Place 100 mL CCMB80 buffer On ice or in the-20 °C freezer for a short time, then on ice.
Centrifuge the culture at3000 rpm, 4°C, 00:20:00 in a flat bottom centrifuge bottle.
Note
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
20m
Discard supernatant by pouring out slowly and then gently using a seropipette to remove the remaining supernatant.
Gently resuspend in 80 mL of ice cold CCMB80 buffer.
Note
Sometimes this is necessarily less than completely gentle. It still works.
Incubate On ice for 00:20:00
Centrifuge again 3000 rpm, 4°C, 00:20:00 and discard supernatant as described above in 15 (then 19 , do not resuspend as per 16 )
Resuspend in 10 mL of ice cold CCMB80 buffer.
Test OD of a mixture of 200 µL SOC media and 50 µL of the resuspended cells.
Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test.
Aliquot to chilled screw top2 mL vials or 50 µL into chilled microtiter plates
Store at-80 °C indefinitely.
Note
Flash freezing does not appear to be necessary. Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles.
Measurement of competence
3m
Transform50 µL of cells with 1 µL of 10 pg/μl pUC19 plasmid in a 1.5 ml or 2 ml microcentrifuge tube.
Note
10 pg/μl or 10-5 μg/μl pUC19 can be made by diluting1 µL of NEB pUC19 plasmid (1 μg/μl, NEB part number N3401S) into 100 mL of TE buffer.
Incubate tubes On ice 00:30:00
Heat shock tubes in a water bath at 42 °C for00:01:00
Incubate On ice for 00:03:00
3m
Add 250 µL SOC media to each tube.
Incubate at 37 °C for 01:00:00 in 2 mL centrifuge tubes, ideally on a lab rotator.
Note
Using 2 mL centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
Ampicillin and kanamycin resistant plasmids require 01:00:00 growth whereas chloramphenicol and tetracycline resistant plasmids may benefit from02:00:00 to increase transformation efficiency and colony yield.
Plate 80 µL on agar plates with the appropriate antibiotic. You can use spreaders or sterile 3.5 mm glass beads to evenly spread out the cells.
Expected result
- Good cells should yield around 100 - 400 colonies
- 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
Protocol references