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

Selective media (LB with 0.25% agar (2,5g/l) was prepared in Petri dishes (85 mm dia.).

1,5 µL of diluted cell suspensions from mid-log-phase cultures (~2×105 cells/µL (OD=0,5)) were applied to the center of the plates, which were dried in air for 15 min, and incubated at 37 °C for 10 h.

Materials

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

Protocol

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.

Comments on the Protocol: As a starter culture you can use an overnight grown 3 ml culture which was picked from a single colony. If possible use also for the starter cultures LB free of NaCl. OD measurements: this is the step which limits quality most. Bacteria are most competent at OD 0.4-0.6 and 0.9. Because it is very difficult to catch them at OD 0.9 every protocol uses OD 0.4-0.6. If the bacteria are over OD 0.6 the competence will be reduced. While harvesting, never let the bacteria warm up again! If you can, work in a cold room on ice. The quality of the competent cells will compensate for the uncomfortable time. Also, from now on it is not necessary to worry about sterility so much. If you get a contamination, it will result in one or two colonies on a plate, so nothing dramatic. While resuspending the pellets, first take a small volume (5ml) to resuspend the pellet in it. Then add the rest. After the last resuspension, measure the OD of a 1:100 dilution. The dilution should have an OD600 of 0.6. If it is higher, you can dilute the suspension more. Before making aliquots, cool the eppis! (you can put the eppis on ice or freeze them in -20°C freezer). Be fast and let a colleague help you. One fills the tubes the other one closes them. In -80°C the cells will stay good at least half a year. Test them after production and retest them if you are not sure if they are still OK. See the transformation protocol for details. At best you can reach 0.5-1.0 x 109 col/µg plasmid. Known Issues: Work fast, clean and cold - you will get good cells. The more practice you get the better the cells will be. If you are not happy with the results, just repeat it and they will be good. If the pulse times are low, the washing was not sufficient.

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