TITLE: Preparation of XL1-Blue competent  cells using MgCl2 and CaCl2
AUTHORS: Rashmi Karki, Monica Rieth

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Preparation of chemically competent Xl1-Blue cells using MgCl</span><span style = "vertical-align:sub;">2</span><span> and CaCl</span><span style = "vertical-align:sub;">2</span></div><div class = "text-block">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.</div><div class = "text-block">This protocol is based on current methods reported in: </div><div class = "text-block">Sambrook, Joseph et al. (2001). Molecular cloning : a laboratory manual. Cold Spring Harbor, N.Y. :Cold Spring Harbor Laboratory Press</div></div>

[STEPS]
?. [Day 1]
Inoculate a 5 mL culture of liquid LB media from a glycerol stock (-80ºC) of XL1-Blue competent cells
?. [Day 1]
Incubate it for 16-20 hours at 37°C at 220-225 rpm
?. [Day 2]
Remove the culture tubes from incubator
?. [Day 2]
Make four dilutions of the culture in liquid broth
?. [Day 2]
Dilution 1: 20μl of the culture  into 980μl LB
?. [Day 2]
Dilution 2: 20μl from dilution 1 into 980μl LB
?. [Day 2]
Dilution 3: 20μl from dilution 2 into 980μl LB
?. [Day 2]
Dilution 4: 20μl from dilution 3 into 980μl LB
?. [Day 2]
Plate 200μl of dilution  4 in LB plate and let it sit for some time until all the culture has been soaked
?. [Day 2]
Keep the  plates inverted  in incubator for 16-20 hours at 37ºC.
?. [Day 3]
Remove the plates from the incubator.
?. [Day 3]
Pick a single bacterial colony (2-3mm in diameter) from a plate (dilution 4) that has been incubated for 16-20 hours at 37ºC.
?. [Day 3]
Transfer into 100 ml sterilized LB broth or SOB medium in a 1-liter flask.
?. [Day 3]
Incubate for 6 hr at 37°C with shaking at 220-250 rpm.
?. [Day 3]
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.
?. [Day 3]
Harvest the cell by arresting the growth when OD600 ̴ 0.45. Immediately put the flask in the ice.
?. [Day 3]
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.
?. [Day 3]
Centrifuge at 2700g (4100 rpm in a Sorvall GSA rotor)or (RC-3B refrigerated centrifuge) for 10 minutes at 4 °C
?. [Day 3]
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
?. [Day 3]
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
?. [Day 3]
Centrifuge at 2700g (4100 rpm in a Sorvall GSA rotor) for 10 minutes at 4 °C
?. [Day 3]
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
?. [Day 3]
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.
?. [Day 3]
Add 140 μl DMSO (cryo-protectant) per 4ml. Swirl gently to mix and let it sit for 15 minutes.
?. [Day 3]
Add additional 140 μl DMSO per 4 ml. Mix gently and return the tube to the icebox.
?. [Day 3]
Transfer and store in aliquots of 100 μl in sterile 1.5 mL Eppendorf tube.
?. [Day 3]
Freeze at -80 °C.