Team:Goettingen/protocol Plasmid Tran

Preparation of E.coli competent cell

Supercompetent E.coli for long term storage

1. Inoculate 4 ml LB medium with the appropriate E. coli strain and incubate the culture overnight at 37°C.
2. Inoculate 500 ml SOB medium with the overnight culture and incubate at 25-30°C until an OD600 of approximately 0.5.
3. Chill the culture for at least 10 min on ice.
During the following steps, the cell suspension should be kept on ice as much as possible.
4. Centrifuge the cell suspension for 10 min at 4,500 to 6,000 rpm at 4°C.
5. Gently resuspend the pellet in 100 ml ice-cold TB buffer.
6. Incubate the cell suspension on ice for 10 min.
7. Centrifuge for 10 min 4000 rpm at 4°C.
8. Gently resuspend the pellet in 18.6 ml ice-cold TB buffer and add 1.4 ml DMSO.
9. Incubate the cell suspension on wet ice for at least 10 min.
10. Aliquot 100 µl of the cell suspension in 1.5 ml tubes.
11. Shock-freeze the cell suspension in liquid nitrogen and store the tubes at -80°C or in liquid nitrogen.
At -80°C the cells will be competent for at least 6 months. In liquid nitrogen they will stay competent indefinitely.

Competent cells with CMMB80 buffer and TOP10 strain

1. Inoculate 250 ml of SOB medium with 1 ml over day culture and incubate at 18°C for approx. 19 hours until it reaches an OD600 of 0.3.
2. Fill an ice bucket halfway with ice. Use the ice to pre-chill as many flat bottom centrifuge bottles as needed.
3. Transfer the culture to the flat bottom centrifuge tubes. Weigh and balance the tubes using a scale.
4. Centrifuge at 3000 g and 4°C for 10 minutes in a flat bottom centrifuge bottle.
5. Decant supernatant into waste and gently resuspend the pellet in 80 ml of ice cold CCMB80 buffer.
     a. Pro tip: add first 40ml to resuspend the cells. When cells are in suspension, add another 40ml CCMB80 buffer for a total volume of 80ml.
     b. Pipette buffer against the wall of the centrifuge bottle to resuspend cells. Do not pipette directly onto the cell pellet!
     c. After pipetting, there will still be some residual cells stuck to the bottom. Swirl the bottles gently to resuspend these remaining cells.
6. Incubate on ice for 20 minutes and centrifuge again at 3000 rpm at 4°C. Decant supernatant.
7. Resuspend cell pellet in 10 ml of ice cold CCMB80 buffer.
8. Measure OD600 and add chilled CCMB80 to yield a final OD of 1.0 - 1.5.
9. Incubate on ice for 20 minutes. Prepare 1.5 mL E-cups, aliquote 100 µl of the cell suspension and freeze them immediately in liquid nitrogen. Store at -80°C.

Fast competent E.coli cells for ready-to-use

1. Inoculate 4 mL LB in a test-tube with a single colony. Grow over night at 37°C.
2. Use 250 µl to inoculate 25 mL of LB in a 250mL bottle the next morning.
3. Shake until an OD600 0.3 - 0.4 is reached.
4. Collect the cells by centrifugation for 5 min at 5000 rpm and 4°C.
5. Discard supernatant and gently resuspend in 10 mL of cold 0.5 M CaCl.
(cells are susceptible to mechanical disruption, so treat them nicely).
7. Incubate on ice for 20 minutes.
8. Centrifuge as described in 4.
9. Discard supernatant and gently resuspend in 0.5(up to 1.5) mL cold 0.5 M CaCl2.
10. Use 50 - 100 µl of the suspension for transformation.

E.coli transformation

- Put 1μl of circular plasmid or all of a ligation reaction of plasmid DNA in a 1.5 mL tube. Gently add ~100 μl of competent cells.
- Incubate for 30 min on ice.
- Heat shock for 2 min @ 42°C. Put back on ice.
- Add 900 μl of LB to tubes. Incubate @ 37°C for 30 min.
- Plate different concentrations (e.g. 50, 100, rest μL) of the cells on LB + antibiotics plates. Incubate them @ 37°C overnight.

Plasmid Isolation from Yeast

1. Collect cells by centrifugation ( room temperature, 4000 rpm, 5 min.) and discard the supernatant.
2. Resuspend the pellet in 250 µl of buffer P1.
3. Add ca. 0.4 g glass beads and break the cells by vortexing vigorously for 5 min.
4. Add 250 µl of buffer P2 and mix immediately.
5. Add 350 µl of buffer N3 and mix thoroughly.
6. Harvest the supernatant (13,000 rpm, 10 min.), transfer it to a new 1.5 ml E-cup.
7. Add 750 µl ice cold isopropanol and incubate 20 min. at 20°C.
8. Harvest the plasmid-DNA (13,000 rpm, 10 min.), discard the supernatant.
9. Wash the plasmid-DNA: Add 500 µl ice cold 70% EtOH and invert 4-6 time and centrifuge (13,000 rpm, 10 min.).
10. Discard supernatant, dry the DNA pellet and resolve the plasmid-DNA in 50 µl sterile distilled water.
11. Measure DNA concentration with Nanodrop.

Yeast transformation “ready-to-use”

- Pick a colony into 5 ml YPAD medium (pH 5.8) and incubate overnight at 30°C with shaking. Until OD600 >1.5.
- Sub-culture 1 ml into 50 ml YPAD and grow at 30°C with shaking for 3.5-4h. Until OD600 0.6-1.2.
- Collect cells by low speed centrifugation (4000 rpm, 5 min, room temperature)
- Discard the medium and resuspend the cells by vortexing in 25 ml dH2O.
- Respin cells (4000 rpm, 5 min, room temperature), discard supernatant and resuspend in 1 ml sterile dH2O.
- Transfer to a 1.5 mL tube and respin at top speed for 10 sec to pellet cells.
- Resuspend in 550 μl 100 mM LiAc pH7.5 and transfer 50 μl aliquots to 11 sterile microcentrifuge tubes.
- Centrifuge for 10 s at top speed to pellet cells, and remove the supernatant.
- To each tube add, in order: 240 μl 50% PEG 4000, 36 μl 1 M LiAc, 10 μl single-stranded DNA (Salmon sperm, Invitrogen).
- 0.5-1 μl plasmid DNA (250-500 ng of each plasmid)
- Resuspend and mix thoroughly by pipetting or vigorous vortexing. Incubate at 42°C for 25 - 45 min.
- Centrifuge cells at low speed (4000 rpm, 10 sec), remove medium and resuspend cell pellet in 1 ml YPAD for 1 h.
- Centrifuge cells at low speed (4000 rpm, 1 min) remove medium and resuspend cell pellet in 200 μl sterile dH2O.
- Spread aliquots onto SC dropout medium (spread gently with spreading bar or using sterile glass beads). Allow to air-dry and incubate at 30°C for 2-3 days for colonies to grow.

Yeast transformation “storage”

1. Grow overnight culture of desired strain in YAPD 25 mL.
2. Inculate 50 mL YPAD with x mL over night culture to OD600 = 0.2-0.3
3. Let the culture grow at 30 °C to OD600 ~0.5-0.7.
4 . Spin down in Falcon tubes for 3 min at 2 krpm and discard the supernatant.
5. Wash with 15 mL sterile water, spin down for 3 min at 2 krpm and discard the supernatant.
6. Wash with 10 mL SORB, spin down for 3 min at 2 krpm and discard the supernatant.
7. Dissolve in 360 µl SORB and add 40 µl SS-DNA, boiled for 10 min at 98 °C and cooled on ice.
AT THIS STEP CELLS CAN BE FROZEN AT -80°C
8. Use 50 µl for transformation of plasmid. Add DNA (maximal 2 µl plasmid DNA/10 µl cells)
9. Add 300 µl LIT-PEG and mix by flicking.
10. Incubate 30 min at 30 °C.
11. Heat shock for 15 min at 42°C.
12. Spin down for 3 min at 2 krpm and discard the supernatant.
13. Wash in 1 mL YPAD, spin down for 3 min at 2 krpm and discard the supernatant.
14. Resuspend the pellet in 100 µl TE buffer.
15. Spread gently on SC plates containing an appropriate selection marker.