Preparation of MS media for Arabidopsis thaliana

For 500 ml of MS (Murashige-Skoog) media measure:

• 5 g of sucrose
• 2.2 g of basal MS salt (Sigma-Aldrich)
• 4 g of agar
• 5 ml of Vitamin stock mix (Gamblor stock solution)
• 500 ml of distilled water

1. Mix all the components, except the agar, in constant agitation.
2. Adjust the pH to 5.7 ± 0.1.
3. Add the agar and mix gently and heat until it reaches 65 – 70 °C. (Do not boil or autoclave)
4. Pour the agar in plates, let them cool and store at 4 °C.

Preparation of Antibiotic stocks

• Cloramphenicol Stock:

1. Weight 10 mg of lyophilized antibiotic for each ml of stock solution.
2. Dissolve in ethanol and mix gently (It is possible to use Vortex)

• Kanamicin Stock:

1. Weight 50 mg of lyophilized antibiotic for each ml of stock solution.
2. Dissolve in distilled water and mix gently (It is possible to use Vortex).

• Ampicilin Stock:

1. Weight 100 mg of liophylized antibiotic for each ml of stock solution.
2. Dissolve in distilled water and mix gently (It is possible to use Vortex).
3. For each 2 ml of media use 1 ul of stock antibiotic.

Therefore the working concentration for each antibiotic (for use in and Agrobacterium tumefaciens and E. coli) will be:
1. Cloramphenicol: 5ul/ml
2. Kanamicin: 25 ul/ml
3. Ampicilin: 50 ul/ml

CaCl2 Competent Cells

1. Streak out frozen glycerol stock of bacterial cells (Top10, DH5α, etc.) onto an LB plate (no antibiotics since these cells do not have a plasmid in them). Work sterile. Grow plate overnight at 37°C.
2. Make sure to autoclave 1 L LB (or your preferred media), 1 L of 100 mM CaCl2, 1 L of 100 mM MgCl2, 100 mL of 85 mM CaCl2, 15% glycerol v/v, 4 centrifuge bottles and caps, lots of microfuge tubes
3. Chill overnight at 4°C 100 mM CaCl2, 100 mM MgCl2, 85 mM CaCl2, 15% glycerol v/v
4. Prepare starter culture of cells
5. Select a single colony of E. coli from fresh LB plate and inoculate a 10 mL starter culture of LB (or your preferred media – no antibiotics). Grow culture at 37°C in shaker overnight.
6. For the next day, inoculate 1 L of LB media with 10 mL starter culture and grow in 37°C shaker.
7. Measure the OD600 every hour, then every 15-20 minutes when the OD getsabove 0.2.
8. When the OD600 reaches 0.35-0.4, immediately put the cells on ice. Chill the culture for 20-30 minutes, swirling occasionally to ensure even cooling. Place centrifuge bottles on ice at this time.
9. (Spin #1) Split the 1 L culture into four parts by pouring about 250 mL into ice cold centrifuge bottles. Harvest the cells by centrifugation at 3000g for 15 minutes at 4°C.
10. Decant the supernatant and gently resuspend each pellet in about 100 mL of ice cold MgCl2. Combine all suspensions into one centrifuge bottle. Make sure to prepare a blank bottle as a balance.
11. (Spin #2) Harvest the cells by centrifugation at 2000g in the refrigerated centrifuge (~3000 rpm) for 15 minutes at 4°C.
12. Decant the supernatant and resuspend the pellet in about 200 mL of ice cold CaCl2. Keep this suspension on ice for at least 20 minutes. Start putting 1.5 mL microfuge tubes on ice if not already chilled.
13. (Spin #3) Harvest the cells by centrifugation at 2000g (~3000 rpm) for 15 minutes at 4°C. At this step, rinse a 50 mL conical tube with ddH2O and chill on ice.
14. Decant the supernatant and resuspend the pellet in ~50 mL of ice cold 85 mM CaCl2, 15% glycerol. Transfer the suspension to the 50 mL conical tube.
15. (Spin #4) Harvest the cells by centrifugation at 1000g (~2100) for 15 minutes at 4°C.
16. Decant the supernatant and resuspend the pellet in 2 mL of ice cold 85 mM CaCl2, 15% glycerol. The final OD600 of the suspended cells should be ~200-250.
17. Aliquot 50 μL into sterile 1.5 mL microfuge tubes and snap freeze with liquid nitrogen. Store frozen cells in the -80°C freezer.

Heat Shock Transformation of E. coli

Note: Never vortex competent cells. Mix cells by gentle shaking.

1. Thaw competent cells on ice. These can be prepared using the CaCl2 protocol.
2. Place 20 ul of cells in a pre-chilled Eppendorf tube.
   • For an Intact Vector: Add 0.5 ul or less to the chilled cells
   • For a Ligation Product: Add 2-3 ul to the chilled cells.
3. Mix gently by flicking the tube.
4. Chill on ice for 10 minutes. (Optional)
5. Heat shock at 42 °C for 50 seconds.
6. Incubate on ice for 2 minutes.
7. Add 200 ul LB, Terrific or SOC medium and recover the cells by shaking at 37 °C.
8. The recovery time varies with the antibiotic selection.
   • Ampicillin: 15-30 minutes
   • Kanamycin or Spectinomycin: 30-60 minutes
   • Chloramphenicol: 60-120 minutes
9. Plate out the cells on selective LB. Use glass beads to spread the cells. The volume of cells plated depends on what is being transformed.
   • For an Intact Vector: High transformation efficiencies are expected. Plating out 10 ul of recovered cells should produce many colonies.
   • For a Ligation Product: Lower transformation efficiencies are expected. Therefore you can plate the entire 200 ul volume of recovered cells.
10. Incubate at 37 °C. Transformants should appear within 8 – 16 hrs.

Transformation by Electroporation

Preparation of Electrocompetent Cells

Competent cells should never be vortexted, as this will cause them to lyse and release salts into the media. Resuspend cells by pipeting up and down with a large pasteur pipet. Once they are chilled, cells should be continuously cold.

1. The night before the transformation, start an overnight culture of cells. 5 ml LB Amp.
2. The day of the transformation, dilute the cells 100X. 100 ml LB Amp.
3. Grow at 30°C for about 90 minutes.
4. Harvest the cells. When the cells reach an OD600 of between 0.6 and 0.8.
5. Split the culture into 2x 50 ml falcon tubes, on ice.
6. Centrifuge at 4 °C for 10 min at 4000 rpm.
7. Wash and combine the cells.
8. Remove the supernatant.
9. Resuspend the cells in 2x 25 ml of ice cold water.
10. Combine the volumes in a single 50 ml falcon tube.
11. Wash the cells 2 more times.
12. Centrifuge at 4 °C for 10 min at 4000 rpm.
13. Resuspend in 50 ml of ice cold water.
14. Repeat.
15. Wash and concentrate the cells for electroporation.
16. Centrifuge at 4 °C for 10 min at 4000 rpm.
17. Resuspend in 1-2 ml of ice cold water.
18. We will use 200 ul of washed cells per transformation.

Dialysis of PCR or Digestion Products

1. DNA for electroporation must be free of salts to avoid arcing.
2. Float a filter in a Petri dish filled with water.
3. Millipore membrane filter 0.025 uM.
4. Pipet one drop of PCR product onto the filter.
5. 200 ng is needed per transformation.
6. 20 - 100 ul fits well on one filter.
7. Collect the drop after 30 - 45 minutes.
8. The volume will change, but the DNA is not lost.

Plasmid purification by Miniprep

1. Centrifuge an eppendorf tube of 1.5ml with L. Plantarum culture at 12000 rpm in the mini-spin for 1 minute, two times.
2. Discard the supernatant and resuspend the tube’s content with more L. Plantarum culture in MRS broth.
3. Centrifuge the eppendorf tube for 15min at 6000 rpm.
4. Discard the supernatant.
5. Resuspend the pellet in 250ml Resuspension buffer.
6. Add 250ml of Lysis Buffer.
7. Gently mix the tube carefully inverting it 5 times carefully.
8. Add and mix softly 350ml of Precipitation Buffer, inverting the tube.
9. Centrifuge it at 12000 rpm for 10 minutes.
10. Transfer the supernatant into a spin column inside a washtube.
11. Centrifuge it at 12000 rpm for a minute.
12. Discard the supernatant and add 500 ml of Wash Buffer with ethanol (w10) to the column.
13. Incubate it for one minute at room temperature.
14. Centrifuge the column at 12000 rpm for 1 minute.
15. Discard the liquid from the washtube and place the column inside the tube.
16. Add 700ml of Wash Buffer W9 with ethanol to the column.
17. Centrifuge the column with the washtube at 12000 rpm for 1 minute.
18. Discard the liquid from the washtube.
19. Centrifuge the column with the washtube at 12000 rpm for 1 minute.
20. Discard the liquid from the washtube.
21. Place the column inside an eppendorf tube of 1.5ml.
22. Add 75ml of preheated TE Buffer at the center of the column.
23. (Warm the TE Buffer previously in water bath at 65⁰c-70⁰c for 3 minutes).
24. Incubate the column for 1 minute at room temperature.
25. The column was centrifuged at 12000 rpm for 2 minutes.
26. (The eppendorf tube contains the purified plasmid).

Plasmid purification by Midiprep

1. Harvest the cells by centrifugation for 10 min at 12000 rpm in the mini-spin. Discard the supernatant.
2. Resuspend the pelleted cells in 2 mL of Resuspension Solution auditioned with RNase solution. The bacterial pellet should be resuspended by vortexing or pipetting up and down until no cell clumps remain.
3. Add 2 mL of Lysis Solution and mix gently by inverting the tube 4-6 times until the solution becomes viscous and slightly clear. Incubate for 3 min at room temperature.
4. Add 0.5 mL of the Endotoxin Binding Reagent. Mix immediately by inverting the tube 5-8 times.
5. Incubate for 5 min at room temperature. Note. After the addition of the Neutralization Solution and Endotoxin Binding Reagent it is important to mix gently, but thoroughly, to avoid localized precipitation of bacterial cell debris. The neutralized bacterial lysate should appear cloudy and contain white precipitate.
6. Add 3 mL of 96% ethanol. Mix immediately by inverting the tube 5-6 times.
7. Centrifuge for 10 min at 4,000-5,000 rpm to pellet cell debris and chromosomal DNA.
8. Transfer the supernatant to a 15 mL tube (not provided) by decanting or pipetting. Avoid disturbing or transferring the white precipitate.
9. Add 3 mL of 96% ethanol. Mix immediately by inverting the tube 5-6 times.
10. Transfer part of the sample (~ 5.5 mL) to the supplied column pre-assembled with a collection tube (15 mL). Be careful not to overfill the column. Centrifuge for 3 min 10000 rpm in the mini-spin. Discard the flow-through and place the column back into the same collection tube.
11. Repeat the last step to process any remaining lysate through the purification column.
12. Add 4 mL of Wash Solution I (diluted with isopropanol) to the purification column. Centrifuge for 2 min. at 4000 rpm in a swinging bucket rotor. Discard the flow-through and place the column back into the same collection tube.
13. Add 4 mL of Wash Solution II (diluted with ethanol) to the purification 6 column. Centrifuge for 2 min. at 5000 rpm. Discard the flow-through and place the column back into the same collection tube.
14. Repeat the column wash with Wash Solution II
15. Centrifuge for 5 min at 3,000 × g in a swinging bucket rotor to remove residual wash solution. Discard the collection tube containing the flow-through.
16. Transfer the column into a fresh 15 mL collection tube (provided). Add 0.35 mL of the Elution Buffer to the centre of the purification column membrane. Incubate for 2 min at room temperature and centrifuge for 5 min at 5000 rpm to elute plasmid DNA.
17. Discard the purification column. Use the purified plasmid DNA in downstream applications or store DNA at -20°C.

Colony PCR

1. Pick a single colony into 30ul of nuclease-free H20. (Fresh colonies grown that day work best, but they can also come from 4 C).
2. Boil for 10 minutes at 100C.
3. Centrifuge (find G) for 1 min. 1ul of this can be used directly for PCR. Best if used directly, but can also be stored at 4C for a few days.

PCR Reaction

Keep all the reagents at 4C while preparing the mixture. Pre-heat the thermocycler to 95C and transfer your reaction directly from 4 C.

Thermocycler Protocol: NEB Quick-Load

Preparation of LB liquid broth

LB is our standard rich media. Directions are on the bottle.

1. Add 12.5g of LB broth powder to a 1L glass bottle.
2. Add 500ml of osmotic water.
3. Autoclave

Preparation of LB Agar

LB agar is our standard rich media for plating. Directions are taken from the bottle.

1. Add 20 g of LB agar powder to a 1 L glass bottle
2. Add 500 ml of osmotic water.
3. Autoclave

Making LB Agar plates

1. Melt LBA in microwave (~8 min).
2. Don’t burn yourself. Most Antibiotics are thermo sensitive so allow agar to cool to approximately 56º C before adding antibiotics.
3. Add antibiotics if required
4. Add 15-25 ml of LBA into each plate. You can get more plates with less agar in each plate, but the plates will desiccate faster and cannot be stored as long.
5. Allow the plates to set.
6. Dry them 45' under the flow hood, with the lid open.

Gel Electrophoresis

Standard 1% agarose gel

Agarose gels are commonly used in concentrations between 0.7 to 2% depending on the size of bands needed to be separated. Simply adjust the amount of starting agarose to %g/100mL TAE e.g. 2g/100mL will give 2%.

1. Measure out 1g of agarose
2. Pour agarose powder into a microwavable flask along with 100mL of 1xTAE
3. Microwave for 1-3mins (until the agarose has dissolved completely and there is a nice rolling boil). Caution HOT! Be careful stirring, eruptive boiling can occur.
4. Let agarose solution cool down for 5min.
5. Pour the agarose into a gel tray with the suitable well comb in place (pour slowly to avoid bubbles which will disrupt the gel).
6. Place newly poured gel at 4°C for 10-15 minutes or let sit at room temperature for 20-30 minutes, until the gel has completely solidified.
7. Once solidified, remove the comb and place the gel into the electrophoresis unit (gel box).
8. Fill the gel box with 1xTAE buffer.
9. Carefully load GeneRuler 100bp Plus DNA weight ladder into the first lane of the gel.
10. Carefully load your samples into the additional wells of the gel.
11. Run the gel at 50-150V until the dye line is approximately 75-80% of the way down the gel.
12. Turn off the power, disconnect the electrodes from the power source and then carefully remove the gel from the gel box.
13. Place the gel into a container filled with 100ml of 1xTAE running buffer and 5μL of EtBr for 5 minutes.
14. Use any device that has UV light to visualize the DNA fragments.