General Protocols

Agarose Gel

1. Weigh 1 g of agarose
2. Add 100 mL of TAE 1X
3. Heat up until the solution is homogeneous, avoiding boiling. If it boils, move away from the heat until it “calms down” and put it back on the heat until the agarose is completely dissolved
4. While heating, prepare the bed in which the gel will polymerize. Make sure that it is well balanced and tight
5. When homogeneous, add 10 µL of SYBR SAFE DNA Gel Stain to the solution and mix well
6. Pour the solution into the bed and clear all its bubbles with a pipette tip. Add in the comb and make sure it is secure
7. Mix the samples with loading dye in a 5:1 ratio. Put the samples into the wells, as well as 5 µL of DNA ladder into the first well

LB Medium (1 litre liquid)

• 10 g tryptone
• 10 g NaCl
• 5 g yeast extract
• Water
• For selective medium, supplement with antibiotic as appropriate (kanamycin 50 µg/ mL and 100 µg/mL for chloramphenicol or ampicillin)

LB Medium (1 litre solid / 50 plates)

• 15 g agar agar
• 10 g tryptone
• 10 g NaCl
• 5 g yeast extract
• Water
• For selective medium, supplement with antibiotic as appropriate (kanamycin 50 µg/ mL and 100 µg/mL for chloramphenicol or ampicillin)

Chemically Competent Cells: CaCl2 Method

1. Inoculate a single colony of appropriate cells into 10ml LB. Add antibiotic if needed and culture o/n at 37°C, 220rpm
2. Inoculate 100ml LB with 1 ml o/n culture
3. Incubate at 37 °C, 220rpm until OD600 = 0.3-0.6 (approx. 2 hrs)
4. Transfer to 2 x 50 ml Falcon and leave on ice for 30 mins
5. Centrifuge at 400 x g, 5 mins, 4 °C
6. Resuspend pellet GENTLY in 25 ml ice cold 0.1 M MgCl2
7. Incubate on ice for 30 min
8. Centrifuge at 4000 xg, 5 min, 4 °C
9. Resuspend pellet GENTLY in 25 ml ice cold 0.1 M CaCl2
10. Incubate on ice for 30 min
11. Centrifuge at 4000 xg, 5 min, 4 °C
12. Resuspend pellet GENTLY in 1.25 ml ice cold CaCl2/Glycerol solution (1.7 ml 0.1 M CaCl2, 0.3 ml 100 % glycerol)
13. Aliquot 100ul and flash freeze on dry ice. Store at – 80 °C

Chemical Transformation by Heat Shock

1. Place a small glass bottle with sterile SOC (10 ml) in the 37 °C incubator to prewarm. Also prewarm at this point the plates for spreading out the transformation
2. Thaw on ice one eppi (200 µl) of self-made competent Top10 cells per ligation to be transformed (Keep the competent cells as much as possible on ice!)
3. Add the entire ligation (20 µl) to the cells (or 0.1 to 1 µl for plasmids to be re-transformed) and mix by tapping gently. Do not mix cells by pipetting, their membrane is fragile and shear force can kill cells at this point!
4. Incubate on ice for 30 minutes (if possible, mix once or twice by gently tapping again)
5. Switch on the water bath immediately after adding the DNA to the cells and set to 42 °C (check temperature with a thermometer, sometimes the setting needs to be adjusted to achieve measured 42 °C. In our lab, set to 39.5 °C to achieve 42 °C!)
6. Heat shock the cells by incubating the eppis for 60 seconds in the 42 °C water bath. Do not mix or shake!
7. Remove the eppis from the 42 °C bath and quickly place on ice, incubate for 2 min
8. Add 500 μL of pre-warmed SOC medium (SOC is a rich medium; use proper sterile technique to avoid contamination)
9. Secure the eppis in a small microcentrifuge rack with tape and place in the 37°C shaking incubator for 60- 90 min (the rack should stick down with its short side so that the eppis lie parallel to the platform for maximum aeration). Warning: if you label only the lids, the tape will remove the labelling when you strip it off after incubation. Label the side of the Eppendorf and secure the label with masking tape!
10. Plate 50- 100 µl of the cells on one selective prewarmed plate. Spin down the remainder of the cells at 3800 rpm for 3 min at RT and pour off the SN until about 50 µl of liquid remain in the tube. Resuspend the pellet in this remaining liquid by pipetting up and down with a 200 µl pipette and plate the entire resuspension onto a second plate
11. Wrap the plates in parafilm and incubate upside down at 37°C o.n.

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