ElectrocompetentCells.doc Page 1 10/2/2018
PREPARATION OF ELECTROCOMPETENT MC1061 CELLS
This protocol illustrates a preparation of a large volume of electrocompetent cells for library construction. Processing consists of growing cells to mid log phase, centrifuging them multiple times in very weak buffer and 10% glycerol to reduce the conductivity, and snap-freezing them in dry ice. The final processed cells are put up in two volumes: 200 µl for actual library construction, and 22 µl for small-scale ligations (e.g., test ligation, cloning constructs, etc.).
PRELIMINARY PREPARATIONS
1. If needed, make 3 liter of L broth. For one liter:
30 g Bacto-tryptone
15 g yeast extract
15 g NaCl
Dissolve in 3 liter water in a 4-liter beaker
Adjust pH to 7.0 with NaOH
2. Measure 750 ml into four 1-liter plastic bottles and autoclave; when cool, pour each into a 2.8-liter Fernbach culture flask autoclaved with a fresh cotton plug.
3. Prepare in advance as follows:
Autoclave, and pre-cool in the refrigerator:
200 ml 10% v/v glycerol
2 liter 1 mM HEPES (pH 7.0)
Precool the SLA1500[1]and FiberLite[2]rotors
Pre-chill in refrigerator:
12 sterile 250-ml centrifuge bottles
4 50-ml centrifuge tubes (or OakRidge tubes if FiberLite rotor not available)
Prelabeled tubes (500-µl Ep tubes OK):
15 tubes labeled
MC1061 EC
Batch 1
200 µl
YOUR REFERENCE
12 tubes labeled
MC1061 EC
Batch 1
22 µl
YOUR REFERENCE
15 tubes labeled
MC1061 EC
Batch 2
200 µl
YOUR REFERENCE
12 tubes labeled
MC1061 EC
Batch 2
22 µl
YOUR REFERENCE
Pre-chill in –80º deepfreeze: an empty pre-labeled container to receive the tubes of frozen electrocompetent cells at step 17 (container can’t be labeled after it has been chilled)
PREPARATION OF FROZEN ELECTRO-COMPETENT MC1061 CELLS
NOTE: This procedure was done twice, staggered by 1 day; the first procedure yielded Batch 1, the second Batch 2. The inoculation step 4 will be done on Day 1 and then again on Day 2. Steps 5–17 will be done on Day 2 (tubes labeled Batch 1) and then again on Day 3 (Batch 2).
4. In a 1 × 3.5 inch culture tube (or other suitable sterile culture vessel) inoculate 6 ml NZY with an MC1061 glycerol stock. Allow to grow in 37º shaker-incubator overnight.
5. Inoculate two flasks from step 2 with 2.5 ml overnight culture previous step. Grow with vigorous shaking to an OD600 of 0.4 as measured in Spectronic 20 colorimeter (this is equivalent to OD600 ~0.4 on our regular spectrophotometer).
NOTE: During the incubation, prepare for processing cells and dispensing them in the cold room:
Pipetters
Sterile pipette tips
Rack with the pre-labeled tubes from step 3
As cultures are nearing the target OD, prepare the pulverized dry ice and bring that to the cold room too.
6. When the target OD600 of 0.4 is reached at the previous step, chill both flasks for 15 min in a large pan of ice-water (not just ice) with frequent swirling. Meanwhile, prepare the pre-chilled centrifuge bottles, pulverized dry ice and pre-chilled BD and 1.5 ml Ep tubes to be used below.
NOTE: Cells should remain as cold as possible during steps 7–14. Keep a large bucket of ice on hand to continually chill the suspensions. Work as quickly as possible, but treat the cells as gently as possible.
7. Pour one of the Fernbach flasks into the other and mix gently; use the combined culture to fill six pre-chilled 250-ml centrifuge bottles all the way to the shoulder (there will be some culture left over—it will just be discarded); centrifuge bottles in pre-cooled SLA1500 rotor at 2000 rpm for 15 min at 4º.
8. Carefully pour off the supernatant (don’t worry if some is left behind).
9. Gently resuspend cells in each bottle in 150 ml ice-cold ice-cold 1 mM HEPES; collect cells by centrifugation as in steps 7–8.
10. Gently resuspend the cells in each bottle in 12 ml ice-cold 1 mM HEPES; pool the resuspended cells in a single bottle (theoretically 72 ml total but probably more like 90 ml in practice); then divide the suspension evenly into two pre-chilled 50-ml tubes. Centrifuge at 2500 rpm for 10 min at 4º in the FiberLite rotor. Gently pour off supernatants.
11. Add ice-cold 10% glycerol to each tube to a total volume of 22.5 ml and gently resuspend cells; pour the contents of one tube into the other.
12. Centrifuge the 50-ml tube at 2500 rpm for 10 min at 4º in FiberLite rotor.
13. Gently pour and aspirate off the supernatant.
14. Gently resuspend cells in 750 µl ice-cold 10% glycerol; the total volume of the suspension in 10% glycerol will be 1.8–3.6 ml. Take in ice bucket to the cold room for next step.
15. In the cold room, deliver aliquots to the bottoms of the appropriate pre-chilled tubes (Batch 1 or 2) as follows:
22 µl into six of the BD tubes labeled “22 µl”
200 µl into as many of the tubes labeled “200 µl” until there’s not enough suspension to make another full 200-µl tube
22 µl into the BD tubes labeled “22 ul” until there’s not enough suspension to make another full 22-µl tube
Close caps.
16. To freeze, pick up tubes one at a time with fingers or a long forceps and submerge for a few moments, right-side-up in crushed dry ice. Leave the tubes in the dry ice after they’re frozen until the next step. Here is a table of the number of 200- and 22-µl tubes in a typical procedure:
BATCH / Number of 200-µl tubes / Number of 22-µl tubes / Approx vol left over / Total volume1 / 15 / 12 / ~300 µl / 3.56 ml
2 / 15 / 11 / 0 / 3.24 ml
17. Take the dry ice with the frozen tubes to the –80º freezer. Working as rapidly as possible, transfer the tubes from the dry ice to the pre-labeled container from step 3. Store the tubes in the –80º freezer until use.
TESTING EFFICIENCY
18. Set up the electroporator at 1250 V and following supplies:
Rack with two labeled 15-ml tubes with 2 ml SOC with 0.2 µg/ml tetracycline
Sterile transfer pipettes in the 15-ml tubes
5-µg/ml fUSE55 electroporation standard in TE or EB (10 mM Tris.HCl pH 8.5)
Space in a shaker-incubator for strapping the rack in
Ice bucket
Two 1-mm cuvettes on ice containing 55 µl 20% glycerol underlay (6.9 ml water, 2 ml glycerol, 1 ml TE pH 8, 100 µl 10-µM phenol red)
eight NZY/Tet plates labeled Batch1/10-2–Batch1/10-5 and Batch2/10-2–Batch2/10-5
19. Remove a single 22-µl tube of electroporation cells from each batch previous step and put them on ice. Immediately do two duplicate electroporations (Batch1 and Batch2) as follows:
Pipette 1 µl (5 ng) of the electroporation standard (on ice; see previous step) into a tube of electrocompetent cells on ice, stirring with pipette tip; if necessary, thaw gently between finger tips.
Leave tube on ice 30 sec.
Carefully layer 18 µl (use regular yellow tip) on top of the red 20% glycerol underlayin a cuvette, being very careful to avoid bubbles.
Zap
Using the transfer pipettes, draw up the SOC in the appropriate 15-ml tube, use it to resuspend the zapped cells by vigorously pumping up and down a few times, and transfer the resuspended cells back into the 15-ml tube (don’t worry about the small volume that remains inaccessible in the cuvette).
Shake the 15-ml tubes at 37º for 45 min.
20. Label eight dilution tubes Batch1/10-2–Batch1/10-5 and Batch2/10-2–Batch2/10-5; into each pipette 450 µl SOC. In these tubes make 10-2–10-5 dilutions of the cells in the two 15-ml tubes by passing 4.55 µl (for the 10-2 dilutions) or 50 µl (for the other dilutions). Plate 200 l of each tube on the correspondingly labeled NZY/Tet plate step 18. Incubate plates overnight in the 37o incubator and count colonies next day. Calculate efficiency as follows:
number of colonies on plate × 2000 × dilution factor = efficiency (colonies/µg)
Dilution / Colonies Batch1 / Colonies Batch210-2 / 830 / ~3500
10-3 / 86 / 412
10-4 / 7 / 48
10-5 / 1 / 2
Efficiency / 1.72 × 108 / 9.6 × 108
[1] Sorvall GSA rotor can be substituted; the advantage of the SLA1500 is its light weight, making acceleration and deceleration much quicker—a boon for limiting damage to cells during processing.
[2] This excellent rotor can centrifuge ordinary disposable screw-cap 50-ml tubes at 15,000 rpm(n this application, though, the rotor will be used only at low speeds). It’s also lightweight, so it accelerates and decelerates very quickly—a boon for limiting damage to cells during processing. The 50-ml disposable screw-cap tubes are superior to round-bottom OakRidge tubes because the cell pellet is at the angle corner rather than the bottom tip of the tube, making it easier to remove most of the supernatant without disturbing the pellet. Nevertheless, the FiberLite rotor isn’t essential: we successfully used an ordinary Sorvall SS34 rotor with OakRidge tubes for years.