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 50-100 µl 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
-
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
Add 5 volumes Buffer PB to 1 volume of the PCR reaction ad mix. If the color of the mixture is orange or volet, add 10 µl 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow
Place a QIAquick column in a provided 2 ml collection tube
To bind DNA, apply the sample to the QIAquick column and centrifuge for 30-60 s. Discard the flow-through and place the QIAquick column back in the same tube
To wash, add 0.75 ml of Buffer PE to the QIAquick column and centrifuge for 30-60 s. Discard flow-through and place the QIAquick column back in the same tube
Centrigue the QIAquick column once more in the provided 2 ml colletion tube for 1 min to remove residual wash buffer
Place each QIAquick column in a clean Eppendorf tube
To elute DNA, add 50 µl Buffer EB (10 mM Tris-Hcl, pH 8.5) or water (pH 7.0-8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge
If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
Excise the DNA fragment from the agarose gel with a clean, sharp scalpel
Weigh te gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg ~ 100 µl). The maxiumum amount of gel per spin column is 400 mg. For >2% agarose gels, add 6 volumes Vuffer QG
Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the
tube every 2–3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10 µl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow
Add 1 gel volume isopropanol to the sample and mix
Place a QIAquick spin column in a provided 2 ml collection tube. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min until all the samples have passed through the column. Discard flow-through and place the QIAquick column back into the same tube. For sample volumes of >80 µl, load and spin again
To wash, add 750 µl Buffer PE to QIAquick column and centrifuge for 1 min. Discard flow-through and place the QIAquick column back into the same tube. Note: If the DNA will be used for salt-sensitive applications (e.g., sequencing, bluntended ligation), let the column stand 2–5 min after addition of Buffer PE. Centrifuge the QIAquick column in the provided 2 ml collection tube for 1 min to remove residual wash buffer.
Place QIAquick column into a clean 1.5 ml microcentrifuge tube
To elute DNA, add 50 µl Buffer EB (10 mM Tris-Hcl, pH 8.5) or water (pH 7.0-8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 µl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge
If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel
Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless.
Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30–60 s and discard the flow-through.
Wash the QIAprep spin column by adding 0.75 ml Buffer PE. Centrifuge for 30–60 s and discard the flow-through. Transfer the QIAprep spin column to the collection tube.
Centrifuge for 1 min to remove residual wash buffer.
Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of the QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
At least 100 μl sample (if you have 20 μl ligation, add 80 μl Mili-Q)
SpeedVac
Procedure
Warning: phenol can cause severe burns to the eyes and the skin. Chloroform is a volatile liquid that affects the central nervous system and a suspected human carcinogenic. It is very volatile and can cause damage by inhalation, skin and eyes absorption, and ingestion. Therefore, perform the whole procedure in a chemical fume hood and wear nitrile gloves (if accidental contact occurs, remove and discard contaminated gloves immediately)
Add one volume of phenol:chloroform:isoamyl alcohol to the sample.
Vortex vigorously for 1 minute.
Spin at 10.000g for 5 minutes. (to separate organic and aqueous phases)
Place the top aqueous solution in a new eppendorf tube and discard the organic bottom phase.
Note: Be careful do not pick up any of the phenol:chloroform:isoamyl alcohol phase.
Repeat the above steps two times.
Now, you can concentrate the DNA sample by using SpeedVac or ethanol precipitation.
Source
We received this protocol from our supervisor Patricia.
Ethanol DNA precipitation
Materials
100% cold ethanol
70% cold ethanol
Eppendorf centrifuge
Sodium acetate (3M) (pH 5.2)
MiliQ water or elution buffer
Eppendorf tubes
At least 100 μl sample (if you have 20 μl ligation, add 80 μl Mili-Q)
SpeedVac
Optional: glycogen
Procedure
Add 0.1x volume of sodium acetate and mix (no vortex)
Optional trick: add 1 μl of glycogen. Glycogen will co-precipate with the DNA and therefore, it will form a visible pellet and incease the recovery of DNA. It van be useful when you have small quantities of DNA.
Add 2.5x volume of 100% cold ethanol and mix (no vortex).
Incubate at -80 °C for at least 2 hours.
Spin at top speed for 15 minutes at 4°C.
Remove the supernatant carefully without touching the pellet.
Spin for some seconds and remove again the excess of supernatant.
Wash by adding 150 μl of 70% cold ethanol by carefully sliding the ethanol along the wall of the tube.
Spin at top speed for 5 minutes at 4°C.
Repeat the ethanol wash from step 8 twice.
Dry with SpeedVac.
Resuspend in the appropriate volume of MiliQ water or elution buffer.
Source
We received this protocol from our supervisor Patricia.
