Chemically Competent Cell Preparation [Calcium Chloride Method]

Harry Akligoh, Jennifer Molloy

Published: 2022-03-30 DOI: 10.17504/protocols.io.261ge4k8dv47/v1

Abstract

This protocol is a low-cost approach for preparing competent cells using 0.1M CaCl₂solution which is useful in cases where commercial competent cells are unaffordable, it is challenging to receive products on dry ice or you do not have an ultra low temperature freezer.

Before start

Disinfect your bench and pipettes using bleach or 70% isopropanol solution

Steps

Overnight plating of E. coli

Plate an innoculum of Escherichia coli (e.g. BL21(DE3), JM109) glycerol stock on LB agar without antibiotics.

Citation

Addgene Streaking and Isolating Bacteria on an LB Agar Plate https://www.addgene.org/protocols/streak-plate/

Note

There are no antibiotics in the culture, so it will be prone to contamination. Use good aseptic technique at all times!

Citation

Source: CDC Public Health Image Library via Wikimedia Commons, in the Public Domain

#尊敬的用户，由于网络监管政策的限制，部分内容暂时无法在本网站直接浏览。我们已经为您准备了相关原始数据和链接，感谢您的理解与支持。
https://lh4.googleusercontent.com/TJ97LJI13MxQJ_9NLUMYjKLZq0Ff2-wuOSNXrubc3McfBK7nx44sz5NVgFj3ncpEWfnFg1GyUv3pMWvZlk6GBv6yPOnuy8XdOB5-v1-5BGqaPpPuaC6fC4-RA3c9Zoim8dLa_Zk42HM

Incubate the plate37°C 2h 0m 0s

Note

Invert the plate(s) downward when incubating overnight. This is to prevent condensed water in the plates flooding the LB agar and smearing your growing bacteria.

Overnight culture

Pick and transfer a discrete single colony of E. coli into 10 ml LB broth using a sterile innoculation loop or toothpick.

Incubate the culture overnight in a shaker incubator at140rpm

Harvesting cells and Washing of Cells

Sub-culture the overnight culture by adding 500µL into 50mL LB broth (i.e. a 1:100 diution) and shake at 140rpm

Monitor OD₆₀₀ every 30 min using a photometer or McFarland Standards until OD₆₀₀= 0.3

Note

Take particular care when aspirating the culture that it does not get contaminated as your media does not contain antibiotics. Aspirate over a Bunsen burner or in a microbiological safety cabinet or use other measures to ensure excellent aseptic technique.

Keep the culture On ice and only mix gently while harvesting the cells, avoid vigorous shaking.

7.1.

Pipette 1mL culture into a 1.5mL microcentrifuge tube and centrifuge at 8000rpm.

Note

If you have a centrifuge that can fit larger tubes, this will speed up the process and avoid multiple steps of pipetting and centrifugation.It is important to keep the cells close to °C during this step and all subsequent steps. If you do not have a refrigerated centrifuge, you can try:putting the rotor in the fridge or freezer prior to useputting the whole centrifuge in a cold room or walk-in fridgekeeping centrifugation time to the minimum needed to form a pellet that is compact enough not to break apart while you discard the supernatant.

After each centrifugation, discard the supernatant making sure not to dislodge the cell pellets and repeat previous step as needed to harvest more cells. If using 1.5mL tubes, we recommend repeating 3-5 times.

Resuspend the harvested cells in 1mL ice cold 0.1Molarity (M) calcium chloride (CaCl₂) solution.

10.

Incubate On ice for 0h 30m 0s

11.

Centrifuge to pellet the cells at 8000rpm

12.

Finally, gently resuspend the cells in 500µL ice cold 0.1Molarity (M) calcium chloride (CaCl₂) solution.

Note

You can also supplement the CaCl₂solution with 15 % v/v glycerol if you wish to store the cells at -80°C . They should then remain competent for at least 1 year. If you do not add glycerol, you must use the cells immediately. Competent cells are not generally stable at -20°C

13.

Pipette 50µL into sterile, ice-cold 1.5mL microcentrifuge tubes to use immediately for transformation or for storage at -80°C .

Note

It is good practice to quantify the transformation efficiency of your competent cells each time you make a batch by transforming them with a known amount (say 1 µL of 50 ng/µL) of plasmid that contains a positive selection marker and counting the number of transformant colonies. This will help track any discrepancies and ensure you get consistent results.

Chemically Competent Cell Preparation [Calcium Chloride Method]

Abstract

Before start

Steps

Overnight plating of E. coli

Overnight culture

Harvesting cells and Washing of Cells

推荐阅读