TITLE: RbCl Uber-Competent Cells
AUTHORS: Sricharan Kadimi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A protocol kindly provided to our team by the advisor to the 2018 MIT iGEM team (Dr. Brian Teague). It is modified slightly from that version (made by Stefan Maas in 1997) to fit our needs. Stefan Maas' protocol is similar to the Hanahan, D. protocol from 1983.</div><div class = "text-block">It worked pretty well to make good competent cells in Summer 2018 and we just used it again in Summer 2019.</div></div>

[STEPS]
?. [Day 0]
Take a tube of comercially competent cells and put it on ice (Thermo-Fisher TOP10 preferred, we think). Make sure to use a tube of comercially competent cells to start, or you may have poorer results. Work near a flame to ensure sterility.Using a sterile pipet tip or a flamed and cooled inoculation loop, take a drop of the thawed competent cells and streak gently onto an LB plate. Put the plate upside-down (agar-side up) in the  incubator for 16-18 hours. Discard the tube of competent cells in bacterial waste (or use for a transformation if possible).See the Streaking Cells protocol for more details.
?. [Day 0]
If you work fast, you might be able to pick a chip of ice from the tube quickly, then return the tube to the -80°C freezer. However, we don't recommend this unless you're experienced, since once the competent cells start warming over -80°C, they lose competency.
?. [Day 0]
Autoclave the following materials:  ABC1AmountMaterialComments222L Erlenmeyer flasksFor culturing cells, second one is if you mess up the first time32500mL bottlesFor TfbI42100mL bottlesFor TfbII52Stir barsFor stirring buffers6~2001.5mL tubes in a glass beakerFor aliquoting competent cells (the whole beaker should just be covered in foil)71500mL graduated cylinderFor measuring media81250mL graduated cylinderFor QSing TfbI91100mL graduated cylinderFor QSing TfbII104200mL centrifuge bottlesFor centrifuge steps, have different orange caps from other bottlesFor best results, autoclave glassware 3/4-full with DI (to remove residual detergent that could interfere with bacterial growth or competency). Plasticware should be rinsed with DI 2-3x and then autoclaved on dry cycle. Any containers without caps should be covered securely in aluminum foil. Autoclave tape should be applied if necessary to foil or outside container walls. See the Autoclave protocol for more details.
ABC1AmountMaterialComments222L Erlenmeyer flasksFor culturing cells, second one is if you mess up the first time32500mL bottlesFor TfbI42100mL bottlesFor TfbII52Stir barsFor stirring buffers6~2001.5mL tubes in a glass beakerFor aliquoting competent cells (the whole beaker should just be covered in foil)71500mL graduated cylinderFor measuring media81250mL graduated cylinderFor QSing TfbI91100mL graduated cylinderFor QSing TfbII104200mL centrifuge bottlesFor centrifuge steps, have different orange caps from other bottles
?. [Day 1]
Make sure you take the plates from the incubator in the morning and leave them in the cold room until you are ready to do your overnight culture.Work near a flame to ensure sterility. Using a serological pipet, fill 2-3 14mL round-bottomed culture tubes with  of LB media. For each culture tube, pick a colony from your plate with a p200 tip and drop it into the media. Put the tubes on a rack and put the rack in the  shaker at 250rpm for 16-18 hours.(You should be able to safely use your SOB + 20mM MgCl2  for this step, that might actually be better. However, we've only tried this with LB).Do NOT add any antibiotic to the culture media - the cells are not antibiotic-resistant and should not be either!See the Overnight Culture protocol for more details.
5 ml
?. [Day 2]
Work near a flame to ensure sterility. Add  fresh overnight culture to   medium in 2L flask. (We used LB but 2xYT and NZDT work, SOB + 20mM MgCl2 is optimal).Flask should be 5x the volume of the medium and covered with foil.
800 µl
400 ml
?. [Day 2]
Shake the culture at 250 rpm at for 3-4 hours until at A600 of 0.5-0.7 (for cuvettes of length 1 cm, A600=OD600).Use the spectrophotometer and cuvettes at our bench (in Quartzy hopefully) for checking the OD (it has a cuvette length of 1cm). Make sure to blank with media. Swirl the culture before taking a 1mL aliquot. Do not return the aliquot to the flask. Work near a flame for sterility.
This is the step where you are most likely to mess up! Start checking your culture after about   and then start checking every 10-15 minutes after that (depending on how much the culture grew). Note that while the culture initially takes a while to get to 0.1 or 0.2 OD600 (lag phase), the OD will start shooting up during the log (exponential phase).
?. [Day 2]
While the cells are shaking, load foam racks and plastic tube racks with open tubes in the cold room. (Put some paper towels over them to prevent dust or anything else from getting into them). Also leave the centrifuge bottles, a box of p200 pipette tips, and several 10mL and 25mL serological pipettes in the cold room to chill. Also turn on the large centrifuge next to the 2nd floor cold room and set it to ,  ,  so it can pre-cool.
?. [Day 2]
Also while the cells are shaking, make fresh TfbI and TfbII. Ideally, these should be made fresh, but if necessary, they can be made the day before as well.   ABC1ComponentTfbI (150mL)TfbII (20mL)2Rubidium chloride1.8 g0.024 g3Manganese (II) chloride tetrahydrate2.30 gnone4Calcium chloride0.17g0.17g5Potassium chloride0.44gnone6MOPSnone0.042g7Glycerol22.4 mL (28.4g)3.0 mL (3.78g)Add all dry reagents to the bottle, add glycerol (usually easier just to weigh it) ~3/4 of the autoclaved MilliQ water (in the past, we just used DI, but autoclaved MilliQ is best) needed to QS.Then adjust pH:For TfbI adjust to pH 5.8 with 0.2M acetic acid.         Add slowly! If you add too much then the solution will turn yellow-green and you will have to remake it.For TfbII adjust to pH 6.5 with 1M KOH.        Add slowly! If you add too much then the solution will turn yellow-green and you will have to remake it.Then transfer to graduated cylinder to QS to final volume with autoclaved MilliQ water.For TfbI and TfbII, filter sterilize with a  filter (not sure about the pore size, but it's what we used).
ABC1ComponentTfbI (150mL)TfbII (20mL)2Rubidium chloride1.8 g0.024 g3Manganese (II) chloride tetrahydrate2.30 gnone4Calcium chloride0.17g0.17g5Potassium chloride0.44gnone6MOPSnone0.042g7Glycerol22.4 mL (28.4g)3.0 mL (3.78g)
You may need to let the TfbI stir a long while to get the pH up to 5.8 or higher before you pH adjust back down!
0
?. [Day 2]
Chill cell culture in ice-water bath for 10-15 minutes.
All steps from here on should be done in the cold room. Make sure to keep the cells as cold as possible for the remainder of the protocol - so all containers, buffers, tips, etc should be cold as well!
?. [Day 2]
Separate culture evenly between two chilled 250 mL centrifuge bottles.Centrifuge at  (about 3000x g) at  for .
Make sure that the cap of the centrifuge bottle screws on correctly and tightly! There are some dud caps that could cause your cell culture to spill out into the centrifuge chamber if used. Also check if there might be any autoclaved O-rings that go with the centrifuge bottles, because that would also help prevent spills.
?. [Day 2]
In the cold room, decant medium and drain on a paper towel to allow all traces of medium to drain out.Tap the bottles on paper towels but be careful not to lose any cells (the pellet may start to slide down the side).
?. [Day 2]
Gently resuspend each cell pellet in 4-8 mL ice-cold TfbI (use serological pipette)."Gently" means lightly swirling the flask until all cells are resuspended. Don't shake, poke, or pipet.DO NOT disturb the cell pellet by pipetting back and forth or shaking vigorously. This may take a while, but make sure the pellets are fully resuspended (uniform solution). Pour one of the resuspended pellets into the bottle with the other resuspended pellet and bring the total volume to  (use serological pipette).Incubate on ice in the cold room for .
120 ml
This is usually a good time to go get lunch :)
?. [Day 2]
Centrifuge again at  (about 3000x g) at  for . Make sure to label the tube containing the cells. Then, fill another centrifuge bottle with water as a balance (and label that differently).
?. [Day 2]
In the cold room, decant the buffer and discard. Gently resuspend cells in  ice cold TfbII (this could take 5-10 minutes, similar to before). Aliquot cells into chilled 1.5mL tubes.Aliquots should be 50-200μL (usually ). Chill your pipette tips in the cold room before aliquotting and swirl cells regularly during aliquotting. Try to work quickly!
16 ml
100 µl
?. [Day 2]
Close the caps of all the 1.5mL tubes in the foam rack. Fill a plastic ice bucket with liquid nitrogen (ask for a PI or grad student's help here). Then place the foam rack into the liquid nitrogen for snap freezing. You will know the tubes are frozen when most of the popping/crackling sounds subside.Snap freezing ideally should be done in liquid nitrogen (as detailed above). However, you can also use dry ice if necessary.Make sure you have some (pre-labeled!) empty freezer boxes to transfer the 1.5mL tubes to after snap freezing. Then put the boxes in the  freezer for storage.If necessary, transfer more of the 1.5mL tubes to the emptied foam rack and repeat until all cell aliquots are snap frozen.
?. [Testing Competency]
Make sure to test your cells for transformation efficiency using the iGEM Comptent Cell Test Kit or similar. (BBa_J04450).Refer to the RbCl Cells Transformation protocol for more details.