Protocols

Preparation of competent Bacterial cells

Preparation of the Bacterial culture

1. Recuperate the overnight bacterial culture
2. Determine the OD600 in a 1 mL Tank Spectro, dilute a 100µl of culture in 900µl of distilled LB
3. In 1L Erlenmeyer add 200 ml of LB and an appropriate volume of culture to have an OD600 = 0.1
4. Put the Erlenmeyer in an incubator for about an hour at 37˚C.
5. Test the OD600 for the new culture
6. The OD600 should be between 0.4 and 0.6

All the handling done in a 15cm radius of an open flame for optimal sterility

Preparation of Buffer Tbf1 and Tbf2

Some solution aren’t available directly and are prepared by dissolving the solid compound

Sterilize the flask prepared after weighing in an autoclave. When preparing Tbf1 and Tbf2 should be in proximity of an open flame

Preparation of Competent Bacterial cells

1. Transfer the Bacterial culture in 50 ml Falcon tubes
2. Centrifuge for 10 minutes at 3500 rpm in cold
3. Remove the supernatant then re-suspend the pellet in 20 ml of Tbf1 for 50 ml of bacteria
4. Centrifuge for 5 minutes at 3500 rpm in cold
5. Poll in all bacterial culture in a single tube
6. Remove the supernatant then re-suspend the pellet in 8 ml of Tbf2 for 200 ml of bacteria
7. Allocate 220 µl of competent Bacterial cells in each eppendorf tube
8. Instant freeze the eppendorf tubes in liquid nitrogen
9. Conserve the tubes at -80˚C

NOTE: all handling done in a cold room. To re-suspend the pellet a hard jerking action applied on the tube. Alternatively, put on wheel for 10 minute.

Transformation protocol

Sensitivity test of competent bacterial cells

1. Heat up LB-agar in micro oven at 300Watt for 19 minutes for 400 mL of LB-agar loosen cap before heating to let steam out
2. Transfer 80 mL of LB-agar in to Erlenmeyer one for every antibiotic
3. Pour 20 mL and spread the LB-agar in petri dishes, let it dry out
4. Prepare petri dishes:

25µL of competent strain spread with glass beads in all four antibiotic and one without antibiotic as negative test

Handling is done near an open flame, sterile environment

Preparation of DNA material from iGEM kit

1. Pierce plate at the desired location
2. Hydrate by injecting 10µL of ddH2O
3. Let it soak for 5 minutes until red coloration is highly visible
4. pipet up-down to homogenize the solution
5. Transfer the solution into an eppendorf tube set it on ice

No special conditions needed when manipulating just respect sterility

Transformation of bacterial cells with plasmid/ligation

1. 20 ng of material are transferred in eppendorf tube, excess are conserved at -20˚C
2. Add 100 µL of competent bacterial cells
3. Put on ice for 20 minutes
4. Put tubes in thermomixer at 42˚C for 45 seconds
5. Put on ice for 5 minutes
6. Add 450 µL of LB
7. Incubate tube for 1 hour at 37˚C
8. Centrifuge for 5 minutes at 5000rpm (normal centrifuge will work)
9. Remove 400 µl of supernatant
10. Re-suspend pellet in the 150 µL of remaining medium pipet up-down
11. Spread total volume on petri dish with the appropriate antibiotic
12. Put petri dishes in an incubator overnight at 37˚C

For a negative control repeat procedure without adding plasmid

Handling is done near an open flame, sterile environment

One-step sequence and ligation-independent cloning (SLIC)

Procedures

1. Digest vector with restriction enzyme(s) overnight, and purify the linearized vector with a commercial PCR purification kit. Elute the DNA with elution buffer or 10mM TrisCl, pH 8.0-8.5. Do not elute the DNA with water or TE. Measure the concentration of the vector.

2. Amplify your gene pf interest by PCR using primer with ≥ 15 mer homology extension to the linearized vector end. We usually use 15bp homology for single fragment cloning, and 20 bp homology for multiple fragment cloning. Purify the linearized vector with a commercial PCR purification kit. Elute the DNA with elution buffer or 10 mM TrisCl, pH 8.0-8.5. Do not elute the DNA with water or TE. Measure the concentration of the insert(s).

3 .Mix the linearized vector and insert at a molar ratio of 1:2 in a 1.5 ml tube. An example is shown as follows. (Vector to insert molar ratio of 1:1 to 1:7 works well, but we usually use 1:2 for single fragment cloning 1:2:2 for multiple fragments cloning. An example of 3 fragment cloning is shown below with vector: insert 1: insert 2 molar ratio is 1:2:2 as shown

4 .Add 0.2 µL of T4 DNA polymerase (3 U/µL,NEB) to the mixture and incubate at room temperature for 2.5 min. we found that 0.5µL(1.5 U) of T4 DNA polymerase gives the best result, But 0.2 µL gives more than sufficient number of colonies.

5 . Put the reaction mixture on ice immediately to stop the reaction and incubate on ice for 10 min

6 .Thaw chemically competent E.coli cells on ice for ~ 10 min

7 .For single fragment cloning, gently mix the cells with 1-2 µL of the reaction mixture and incubate the cells on ice for 20 min. for multiple fragments cloning, mix the cells with 3-5 µL of the reactant.

8 .Incubate the cells on ice for 20 min.

9 .Heat shock the cells at 42˚C for 45 sec

10 .Incubate the cells on ice for 2 min.

11 .Add 900 (or 950) µL of LB broth to 100 (or 50) µl of cells and transfer the cells to 15 ml round-bottom tube.

12 .Incubate the cells at 37˚C for 1 hour

13 .Plate the cells on agar plates containing suitable antibiotics (eg.100 µg/mL ampicillin). We usually spread 10 to 20 µL of cells onto an agar plate to get optimal number of colonies per plate for single fragment cloning, and 100 µL per plate for multiple fragments cloning. In case of 10-20 µL spreading, cells needs to be further silted with 80~ 90 µL LB broth for suitable spreading.

14 .Incubate the plates at 37˚ for 16 hr and analyze the colonies