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

Make a liquid culture of a single colony in 1-3 mL salt free LB

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.

Theory

The Gibson assembly, as described by Gibson et al., is a rapid DNA assembly method which assures directionional cloning of fragments in one single reaction. For the Gibson assembly to happen, three essential enzymes are needed : a mesophylic nuclease, a thermophylic ligase and a high fidelity polymerase. For this reaction, we used NEBbuilder. In the first step of this reaction, the exonuclease rappidly cleave off the 5’ DNA ends. These enzymes are then heat inactivated at 50 degrees. In the second step, the complementary overhangs, which have to be put in there upon desiging of the fragments, start to anneal.The polymerase then fills in the gaps. In the final step, the ligases covalently joins both ends. After this, the plasmid should be ready to be transformed. (this text was based on https://www.idtdna.com/pages/docs/default-source/user-guides-and-protocols/gibson-assembly.pdf?sfvrsn=16, as on 13/09/2015)

Add 1 µl DNA to 50 µl electrocompetent cells in an ice-cold cuvette (1 mm)
Electroporate (Eppendorf, 1700 V, 4 msec)
Add 950 µl of SOC solution
Incubate for one hour at 37 °C
Plate this out on pre-warmed plates (37 °C)
J23101, J23106 and J23117 were plated out on chloramphenicol and I13504 was plated out on ampicillin

Digest I13504 (GFP) with XbaI and PstI in the Tango buffer
Digest the promoters J23101, J23106 and J23117 with PstI in buffer O
Load the digested I13504 on a 1.5% agarose gel and visualise it under UV light. Thereafter perform a gel purification of I13504 (GeneJET Gel Extraction Kit - ThermoFisher Scientific)
PCR purify the promoters J23101, J23106 and J23117
Digest the promoters J23101, J23106 and J23117 with FD SpeI in 10x Fast Digest Buffer
Ligate every promoter with I13504 using T4 DNA ligase

Digest with NcoI (cuts 1x in pSB1C3) and XhoI (cuts 1x in GFP) in a Tango buffer
Mix gently and spin down
Put this for 2 hours at 37 °C in a heating block
Gel electrophoresis of the samples in 1.5% agarose gel
Analyse the gel picture and interpret the results

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