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.