Jun 13, 2019
RbCl Uber-Competent Cells
- 1University of Connecticut
- UConn iGEM
Protocol Citation: Sricharan Kadimi 2019. RbCl Uber-Competent Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.3hygj7w
Manuscript citation:
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 in our group and it is working, though open to further improvement/modification
Created: May 30, 2019
Last Modified: June 13, 2019
Protocol Integer ID: 23832
Keywords: Competent Cells
Abstract
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.
It worked pretty well to make good competent cells in Summer 2018 and we just used it again in Summer 2019.
Materials
MATERIALS
MOPSP212121
Manganese(II) chloride tetrahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3634
GlycerolBio Basic Inc.Catalog #GB0232.SIZE.500ml
Rubidium chlorideBio Basic Inc.Catalog #RB0668.SIZE.25g
Potassium acetateMerck Millipore (EMD Millipore)Catalog #1.04820.1000
Calcium ChlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #C4904
Potassium hydroxideMerck MilliporeSigma (Sigma-Aldrich)Catalog #1050121000
Acetic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #695092
Make sure you also have all the glassware/plasticware you need (listed in the first step), as well as:
- SOB media + 20mM MgCl2
- LB plates without antibiotic (2)
- LB media
- Plastic tube racks with foam inside
- Labeled boxes for storing 1.5mL tubes
Day 0
Day 0
Autoclave the following materials:
| Amount | Material | Comments | |
| 2 | 2L Erlenmeyer flasks | For culturing cells, second one is if you mess up the first time | |
| 2 | 500mL bottles | For TfbI | |
| 2 | 100mL bottles | For TfbII | |
| 2 | Stir bars | For stirring buffers | |
| ~200 | 1.5mL tubes in a glass beaker | For aliquoting competent cells (the whole beaker should just be covered in foil) | |
| 1 | 500mL graduated cylinder | For measuring media | |
| 1 | 250mL graduated cylinder | For QSing TfbI | |
| 1 | 100mL graduated cylinder | For QSing TfbII | |
| 4 | 200mL centrifuge bottles | For centrifuge steps, have different orange caps from other bottles |
Make sure all bottles have their caps on loosely!
For 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.
2h
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 37 °C 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.
20m
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 1
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 5 mL 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 37 °C 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.
20m
Day 2
Day 2
Work near a flame to ensure sterility. Add 800 µL fresh overnight culture to 400 mL 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.
5m
Shake the culture at 250 rpm at 37 °C 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.
Note
This is the step where you are most likely to mess up! Start checking your culture after about 02:30:00 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).
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 4 °C , 4000 rpm , 00:10:00 so it can pre-cool.
15m
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.
| Component | TfbI (150mL) | TfbII (20mL) | |
| Rubidium chloride | 1.8 g | 0.024 g | |
| Manganese (II) chloride tetrahydrate | 2.30 g | none | |
| Calcium chloride | 0.17g | 0.17g | |
| Potassium chloride | 0.44g | none | |
| MOPS | none | 0.042g | |
| Glycerol | 22.4 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.
Note
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!
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.
Then transfer to graduated cylinder to QS to final volume with autoclaved MilliQ water.
For TfbI and TfbII, filter sterilize with a 0.4 μm filter (not sure about the pore size, but it's what we used).
1h 30m
Chill cell culture in ice-water bath for 10-15 minutes. 00:10:00
Note
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!
10m
Separate culture evenly between two chilled 250 mL centrifuge bottles.
Note
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.
Centrifuge at 4000 rpm (about 3000x g) at 4 °C for 00:10:00 .
10m
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).
5m
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 120 mL (use serological pipette).
Incubate on ice in the cold room for 02:00:00 .
Note
This is usually a good time to go get lunch :)
2h 10m
Centrifuge again at 4000 rpm (about 3000x g) at 4 °C for 00:10:00 .
Make sure to label the tube containing the cells. Then, fill another centrifuge bottle with water as a balance (and label that differently).
10m
In the cold room, decant the buffer and discard. Gently resuspend cells in 16 mL 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 100 µL ). Chill your pipette tips in the cold room before aliquotting and swirl cells regularly during aliquotting. Try to work quickly!
25m
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 -80 °C 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.
15m
Testing Competency
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.