Difference between revisions of "Team:KU Leuven/Research/Methods"

• 1. Preparation of lysate starting from stock plate of phage
  • Stockplate of E. coli MG1655
  • - O/N culture of E. coli MG1655
  • - Take 500 µl of O/N culture and add P1 (-80°C). Incubate O/N at 37°C
  • - Take single plaques of P1 stock plate and bring it in a sterile eppendorf tube together with 200 µl of MQ
  • - O/N extraction, shaking at 37°C
  • - Add 0.01, 0.1, 1, 10 and 100 µl of extraction to 500 µl of a stationary phase culture of E. coli MG1655. Vortex and bring to a sterile petri dish with LB.
  • - Add LB soft agar with 10 mM MgSO4 and 5 mM CaCl2 in it and incubate at 37°C.
  • - Take plate with best lysis.
  • - Sterilize spoon in bunsen flame, cool down with water and wash with 100% of ethanol.
  • - Put soft agar in syringe (10 ml and G22).
  • - Centrifugation of eppendorf tubes at maximal speed (14 000 rpm) during 10 minutes.
  • - Take 650 µl and bring in new eppendorf tube (total 1300 µl).
  • - Extraction with 30 µl of CHCl3
  • - Vortex heavy!!!
  • - Storage of lysate at 4°C.


• 2. Preparation of lysate of donor strain.
  • - Add to 500 µl aliquots of O/N stationary phase culture of donor strain 0.1, 1, 10 and 100 µl of lysate. First, you have to centrifugate the lysate to be sure the chloroform is at the bottom of the eppendorf tube.
  • - Add LB softagar with 10 mM MgSO4 and 5 mM CaCl2. Incubate at 37°C.
  • - Sterilize spoon in bunsen flame, cool down with water and wash with 100% of ethanol.
  • - Centrifugation of eppendorf tubes at maximal speed (14 000 rpm) during 10 minutes.
  • - Take 650 µl and bring in new eppendorf tube (total 1300 µl).
  • - Extraction with 30 µl of CHCl3
  • - Vortex heavy!!!
  • - Storage of lysate at 4°C.


• 3. Transduction to acceptor strain.
  • - Concentrate 500 µl of O/N stationary phase culture of acceptor strain 5 times in LB with 10 mM MgSO4 and 5 mM CaCl2.
  • - Add 0.1, 1, 10 and 100 µl of the donor strain lysate to 100 µl aliquots of the acceptor strain.
  • - Incubate 30 minutes at 37°C
  • - Plate out on selective medium en incubate overnight
  • -Plate also lysate out on normal LB plate to see if lysate contains no contamination.

Het volledige protocol van 5

Het volledige protocol van 5

Het volledige protocol van 5

Materials

• Single colony of E. coli cells to be transformed
• LB medium
• 0.1 M CaCl2, ice cold
• LB amp plates
• 42 °C water bath
• 0.1 M CaCl2+15% glycerol, sterile

Procedure:

1. Inoculate one colony from LB plate into 2 ml LB liquid medium. Shake at 37 °C overnight. 2. Inoculate 1-ml overnight cell culture into 100 ml LB medium (in a 500 ml flask). Shake vigorously at 37 °C to OD600 ~ 0.25-0.3 (usually it takes about 1.5-hours). 3. Chill the culture on ice for 15 min. Also make sure the 0.1M CaCl2 solution and 0.1M CaCl2 plus 15% glycerol are on ice 4. Centrifuge the cells for 10 min at 3300 g (e.g. 4,000 rpm in the Jouan tabletop centrifuge) at 4 °C. 5. Discard the medium and resuspend the cell pellet in 30-40 ml cold 0.1M CaCl2. 6. Keep the cells on ice for 30 min. 7. Centrifuge the cells as above. 8. Remove the supernatant, and resuspend the cell pellet in 6 ml 0.1 M CaCl2 solution plus 15% glycerol. 9. Pipet 0.4-0.5 ml of the cell suspension into sterile 1.5 ml micro-centrifuge tubes. Freeze these tubes on dry ice and then transfer them to -70 C freezer. Notes: 1. The transformation efficiency is about 1-5x106 prepared with this method. 2. Important: all steps after harvesting the cell should be done on ice (or at 4 °C) 2. The frozen competent cells are stable for 6 months, but once a tube is taken from the freezer and thawed, any unused portion should be discarded. 3. After the competent cells are made, the transformation efficiency should be checked by transformation using plasmid DNA of known concentration /ul DNA when using the competent cells

You will need:

• 1L sterile LB without NaCl (10g tryptone, 5g yeast extract per 1L)
• 500 mL of 10% v/v glycerol
• cold falcon tubes of 50 mL
• cold eppies and pipette tips

• Day 1:
  • Strike your cells on a plate and grow overnight in 37°C.
• Day 2:
  • Pick a single colony from your plate and grow it in 1-3 mL salt free LB overnight in 37°C.
• Day 3:
  • 1. Grow 300-400 mL cells (without salt) in 37°C untill the OD reaches 0.6 (use a starting
  culture).
  • 2. Cool down on ice and from now on perform all the steps in 4°C.
  • 3. Spin the cells down in falcon tubes (3500 g, 20 min, 4°C). Using falcon tubes ensures no detergents present.
  • 4. Resuspend the pellet in 30 mL icecold 10% glycerol (filtered to a disposable bottle to ensure no detergents). Spin down the cells (5000 g, 10 min, 4°C). Repeat this step 3 times.
  • 5. Resuspend the cells in 10% glycerol to obtain a dense pulp (usually not much more than 1.5 mL).
  • 6. Take 50 µL sample and do the electroporation test (without DNA). You should have a pulse of 4-6 msec. If it is shorter, wash the cells once again with 30 mL glycerol.
  • 7. Aliquot the cells (50 µL) and quick-freeze in liquid nitrogen and store at -80°C.
  
  
  

Het volledige protocol van 5