{{{title}}}

{{{toc}}}

Protocols

Preparation of competent Bacterial cells

Preparation of the Bacterial culture

Recuperate the overnight bacterial culture
Determine the OD600 in a 1 ml Tank Spectro, dilute a 100µl of culture in 900µl of distilled LB
In 1L Erlenmeyer add 200 ml of LB and an appropriate volume of culture to have an OD600 = 0.1
Put the Erlenmeyer in an incubator for about an hour at 37˚C.
test the OD600 for the new culture
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

Tbf1 buffer	Total volume 80mL
KAc 1M	2.4 mL
MnCl2 0.5M	8 mL
KCl 1M	8 mL
CaCl₂ 0.1M	8 mL
GlY 80%	15mL
H₂O	38.6 mL

Tbf2 buffer	Total volume 8 mL
NaMOPS 0.2 M	400 µL
CaCl₂ 0.1 M	6 mL
Gly 80%	1.5 mL
KCl 1 M	80 µL
H₂O	500 µL

Some solution aren’t available directly and are prepared from the crystal form

	Volume (mL)	Mass (g)	Molecular weight (g/mol)
MnCl₂ 0.5 M	200	19.791	197.91
KCl 1 M	200	17.91	74.55
NaMOPS 0.2 M	50	2.0926	209.26
KAc 1M	50	4.9	98.15

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

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

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
Transfer 80 mL of LB-agar in to Erlenmeyer one for every antibiotic
Pour 20 mL and spread the LB-agar in petri dishes, let it dry out
Prepare petri dishes:

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

Antibiotic stock	Desired concentration	Volume of antibiotic per mL of LB-agar	Volume in Erlenmeyer
Ampicillin 25mg/mL	100 µg/mL	4 µL/mL	320 µL
Kanamycin 10mg/mL	50 µg/mL	5 µL/mL	400 µL
Tetracycline mg/mL	15 µg/mL	1 µL/mL	80 µL
Chloramphenicol 30 mg/mL	50 µg/mL	1.6 µl/mL	128 µL

Handling is done near an open flame, sterile environment

Preparation of DNA material from iGEM kit

Pierce plate at the desired location
Hydrate by injecting 10µL of ddH2O
Let it soak for 5 minutes until red coloration is highly visible
With an up-down action homogenize the solution
Transfer the solution into an eppendorf set it on ice

No special conditions needed when manipulating just respect sterility

Transformation of bacterial cells with plasmid

2 µL of DNA material is transferred in eppendorf tubes, rest are conserved in -20˚C
Add 100 µL of competent bacterial cells
Put on ice for 20 minutes
Put tubes in thermomixer at 42˚C for 45 seconds
Put on ice for 5 minutes
Add 450 µL of LB
Incubate tube for 1 hour at 37˚C
Centrifuge for 5 minutes at 5000rpm (normal centrifuge will work)
Remove 400 µl of supernatant
Re-suspend by up-down action the 150µL of solution
Spread total volume on petri dish with the appropriate antibiotic
Put petri dishes in an incubator overnight at 37˚C