Grow 5ml overnight culture of cells in LB media, dilute this culture back into 25-50ml of fresh LB media in a 200ml conical flask.
In the morning You should aim to dilute the overnight culture by at least 1/100.
Grow the diluted culture to an OD600 of 0.2 - 0.5. (You will get a very small pellet if you grow 25ml to OD600 0.2)
Put Eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later. If your culture is X ml, you will need X tubes. At this point you should also make sure that your TSS is being cooled (it should be stored at 4°C but if you have just made it fresh then put it in an ice bath).
Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.
All subsequent steps should be carried out at 4°C and the cells should be kept on ice whenever possible.
Centrifuge for 10 minutes at 3000 rpm and 4°C.
Decant supernatant, remove leftover media by carefully pipetting
Resuspend in TSS buffer (10% of original volume), vortex gently
Add 100 μl aliquots to chilled Eppendorfs, flash freeze and store at – 80°C in 200 µl aliquots.
Thaw competent cells on ice. When using multiple aliquots, first pool all cells into a single volume to homonogize the solution. Dispose of unused competent cells. Do not refreeze since reusing thawed cells, will drastically reduce transformation efficiency.
Pipet 50 µl of competent cells into Eppendorf tube for each transformation (labeled, prechilled, in floating rack), don’t forget control tubes
Pipet 1-100 ng of DNA as well as control into tubes and gently mix with tip
incubate on ice for 30 min, tubes may be gently flicked, return to ice ASAP
Meanwhile, for every transformation, add 2 µl of each SOC salt solution into 100 µl 2x SOC stock and ad to 200 µl with sterile MQ. Place the SOC medium on ice till use
Heat shock tubes at 42°C for 30 seconds (precisely)
Incubate on ice for 5 min
Add 200 µl of SOC medium to each transformation
Incubate at 37°C for 1 hours, shaker or rotor recommended
Pipet 20 µl & 200 µl transformation mixture onto petri plates and spread with sterilized spreader or glass beads. Let the plates dry near the flame before placing them in the incubator
Incubate plates upside down overnight (14-18hr) at 37°C
Pick single colonies
Perform Colony PCR to verify
Grow cells & miniprep
Calculate efficiency by counting colonies (expected value: 1.5x10^8 to 6x10^8 cfu/µg DNA)
Plasmid DNA (preferably without salts from buffers. These can be removed by incubating the DNA on a filter, which is floating on MQ for 10 minutes at room temperature
Electrocompetent L. lactis cells: 50 µl per transformation
Mix all elements of the recovery medium (1 ml per transformation) and place it on ice together with the electroporation cuvettes
Thaw competent cells on ice. When using multiple aliquots, first pool all cells into a single volume to homonogize the solution. Dispose of unused competent cells. Do not refreeze since reusing thawed cells, will drastically reduce transformation efficiency.
Pipet 50 µl of competent cells into Eppendorf tube for each transformation (labeled, prechilled, in floating rack), don’t forget control tubes
Add at most 5 µl of plasmid DNA to the competent cells and incubate on ice for 10 minutes
Dry the cuvette, electroporate at 2500 V and carefully add 950 µl recovery medium to the cells. Place the cuvette back on ice and incubate for 10 minutes
Transfer the cell suspension with careful mixing into Eppendorf tubes and incubate for 2 hours at 30°C
Plate 100 µl of the recovered cells onto a selection plate and spin down the remaining cells at 6000 rpm for 5 minutes
Resuspend cell pellet into 50-100µl and plate on a selection plate
Template DNA (colony resuspended in MQ / plasmid DNA)
Taq DNA Polymerase
Nuclease-free water
PCR tubes
Ice
PCR tube rack
Procedure
Suspend a colony in 10 µl sterile MQ
Prepare Mastermix for 10 reactions according to:
Component
220 µl = 10 colonies
Final Concentration
10X Standard Taq (Mg-free) Reaction Buffer
22 µl
1X
25 mM MgCl2
13,2 µl
1.5 mM
10 mM dNTPs
4,4 µl
200 µM
10 µM pJET fw
4,4 µl
0.2 µM (0.05–1 µM, typically 0.1-0.5µM)
10 µM pJET rv
4,4 µl
0.2 µM (0.05–1 µM, typically 0.1-0.5µM)
Taq DNA Polymerase
1,1 µl
1.25 units/50 µl PCR
Nuclease-free water
148,5 µl
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Put 19 µl of mastermix in each reaction tube & add 2 µl suspended colony mixture
Add 2 µl plasmid DNA for positive control and 2 µl MQ for the negative control
Place the tubes in the PCR machine (Taq program)
Once the PCR is done, mix 10 µl of PCR product with 2 µl 6X purple gel loading dye and run it on a gel for 50 minutes at 130 Volts
If the correct products are present in the gel samples, inoculate overnight cultures from the original plates.
Mix 5 ml LB with appropriate antibiotic. Scoop a colony from the plate and drop the tip into the medium. Incubate the tube at 37°C overnight to let the culture grow
