Apr 12, 2020
Preparation of XL1-Blue competent cells using MgCl2 and CaCl2
- 1Southern Illinois University-Edwardsville
- Labyrieth
Protocol Citation: Rashmi Karki, Monica Rieth 2020. Preparation of XL1-Blue competent cells using MgCl2 and CaCl2. protocols.io https://dx.doi.org/10.17504/protocols.io.3trgnm6
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: June 05, 2019
Last Modified: April 12, 2020
Protocol Integer ID: 24145
Keywords: XL1-Blue competent cells, Calcium-Chloride method
Abstract
Preparation of chemically competent Xl1-Blue cells using MgCl2 and CaCl2
The purpose is to prepare batches of chemically competent bacteria for the purposes of subcloning and protein expression. Competent bacteria are able to readily and passively take up foreign DNA due their compromised cell walls as a result of exposure to divalent metal ions.
This protocol is based on current methods reported in:
Sambrook, Joseph et al. (2001). Molecular cloning : a laboratory manual. Cold Spring Harbor, N.Y. :Cold Spring Harbor Laboratory Press
Guidelines
This protocol must be carried out under sterile conditions. Wherever possible working benchtop and spaces should be wiped down with 70% EtOH solution. Sterilized, gloved hands are a must at all times.
Materials
MATERIALS
CaCl2
MgCl2
DMSO
Micropipettes and tips
Eppendorf tubes (1.5 & 2.0 ml)
Paper towels
LB Broth
XL1-Blue cells
sterile culture tubes (17 x 100 mm)
high-speed preparatory centrifuge
Safety warnings
Proper clothing is required as it needs to be done in cold room (4 °C)
Keep the DMSO at room temperature
Wear gloves at all times
Before start
Autoclave 100 ml LB- broth in a 1000 ml flask
Sterilize and pre-chill the 50-mL conical tubes
Prepare 30 ml of ice cold MgCl2-CaCl2 solution (80mM MgCl2, 20 mM CaCl2)
Prepare ice cold 0.1 CaCl2
Pre-warm the LB plates
Day 1
Day 1
Inoculate a 5 mL culture of liquid LB media from a glycerol stock (-80ºC) of XL1-Blue competent cells
Incubate it for 16-20 hours at 37°C at 220-225 rpm
Day 2
Day 2
Remove the culture tubes from incubator
Make four dilutions of the culture in liquid broth
Dilution 1: 20μl of the culture into 980μl LB
Dilution 2: 20μl from dilution 1 into 980μl LB
Dilution 3: 20μl from dilution 2 into 980μl LB
Dilution 4: 20μl from dilution 3 into 980μl LB
Plate 200μl of dilution 4 in LB plate and let it sit for some time until all the culture has been soaked
Keep the plates inverted in incubator for 16-20 hours at 37ºC.
Day 3
Day 3
Remove the plates from the incubator.
Pick a single bacterial colony (2-3mm in diameter) from a plate (dilution 4) that has been incubated for 16-20 hours at 37ºC.
Transfer into 100 ml sterilized LB broth or SOB medium in a 1-liter flask.
Incubate for 6 hr at 37°C with shaking at 220-250 rpm.
Measure OD600 of culture every 15-20 minutes to ensure that the culture does not grow to a higher density. The transformation is efficient at OD600 ̴ 0.45.
Harvest the cell by arresting the growth when OD600 ̴ 0.45. Immediately put the flask in the ice.
Transfer the bacterial cells to pre sterilized and ice-cold 50 ml conical tube. Cool the cultures to 0°C by storing the tubes on the ice for 10 minutes.
Centrifuge at 2700g (4100 rpm in a Sorvall GSA rotor)or (RC-3B refrigerated centrifuge) for 10 minutes at 4 °C
Decant the medium from the cell pellets and keep the tubes in an inverted position over the paper towel for 1 minute to allow the traces of media to drain away
Resuspend the pellets by swirling or gentle vortexing in 30 ml of ice-cold MgCl2-CaCl2 solution(80mM MgCl2, 20 mM CaCl2) and mix until you get rid of all visible chunks
Centrifuge at 2700g (4100 rpm in a Sorvall GSA rotor) for 10 minutes at 4 °C
Decant the medium from the cell pellets and keep the tubes in the inverted position over the paper towel for 1 minute to allow the traces of media to drain away
Resuspend the pellets by swirling or gentle vortexing in 2 ml of ice-cold 0.1 CaCl2 solutions for each 50 ml of the original culture and leave the tubes on ice for 15 minutes.
Add 140 μl DMSO (cryo-protectant) per 4ml. Swirl gently to mix and let it sit for 15 minutes.
Add additional 140 μl DMSO per 4 ml. Mix gently and return the tube to the icebox.
Transfer and store in aliquots of 100 μl in sterile 1.5 mL Eppendorf tube.
Freeze at -80 °C.