Team:KU Leuven/Research/Methods
Methods
P1 transduction
- 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.
- - 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.
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Gibson Assembly
Het volledige protocol van 5
Miniprep
Het volledige protocol van 5
Gel purification
Het volledige protocol van 5
Chemocompetent cells
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Electrocompetent cells
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Transformation
Het volledige protocol van 5