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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 by dissolving the solid compound

	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 each falcon tube
Poll in all bacterial culture in two Falcon tubes with 40 ml each
Centrifuge for 5 minutes at 3500 rpm in cold
Remove the supernatant then re-suspend the pellet in 4 ml of Tbf2 for each falcon tube
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 glass beads 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 25 mg/mL	100 µg/mL	4 µL/mL	320 µL
Kanamycin 10 mg/mL	50 µg/mL	5 µL/mL	400 µL
Tetracycline 15 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
pipet up-down to homogenize the solution
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

20 ng of DNA are transferred in eppendorf tube, excess are conserved at -20˚C
Add 100 µL(50 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 (900 µL) of LB
Incubate tube for 1 hour at 37˚C
Centrifuge for 5 minutes at 5000rpm (normal centrifuge will work)
Remove 400 µl(850 µL) of supernatant
Re-suspend pellet in the 150 µL of remaining medium pipet up-down
Spread total volume on petri dish with the appropriate antibiotic
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

	Stock concentration	Volume added	Final concentration
Lineaeized Vector (eg, 5Kb)	100 ng/µL	1 µL	10 ng/µL
Insert 1 (PCR product eg, 1kb)	40 ng/µL	1 µL	4 ng/µL
Insert 2 (PCR product eg, 1kb)	40 ng/µL	1 µL	4 ng/µL
10X BSA		1 µL	1X
10X NEB Buffer 2		1 µL	1X
H₂O		Up to 10 µL

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

Cloning protocol for IDT sequences

Re-suspending gene fragments

Centrifuge for 5 seconds at 3000g
Add 20 µL of TE to the tube for a final concentration of 10ng/µL
Briefly vortex and centrifuge
Tube can be stored at -20˚C

Digest with the restriction endonucleases

Add the following components

Product	Gene fragment	Vector
DNA	100 ng or 10 µL	600 ng
10X Buffer 2	5 µL	5 µL
Restriction Enzyme	1 µL(each)	1 µL(each)
ddH₂O	33 µL	33 µL

Incubate for 45 minutes at 37˚C
Incubate for 20 minutes at 80˚C

Gene fragment and vector digested separately

Transformation

Repeat protocol as mentioned previously

Polylerase chain reaction (PCR)

Reaction setup

Add the following component at listed below

Component	25 µL reaction	50 µL reaction	100 µL reaction	Final concentration
EconoTaq PLUS GREEN 2X Master Mx	12.5 µL	25 µL	50 µL	1x
10 µM Forward primer	2.5 µL	5 µL	10 µL	1 µM
10 µM reverse primer	2.5 µL	5 µL	10 µL	1 µM
DNA Template (10ng/µL)	Variable	Variable	Variable
ddH₂O	To 2.5 µL	To 50 µL	To 100 µL

Assemble all reaction components on ice and quickly transfer the reactions to thermocycle preheated to 94°C and begin thermocycling

Hint: make a premix of the components mentioned without DNA template and allocate appropriately 12 µL in each tube

Thermocycling conditions for a routine PCR

Step	Temperature	Time
Initial Denaturation	95°C	30 seconds
30 Cycles	95°C 45-68°C 68°C	15-30 seconds 16-60 seconds 1 minute/Kb
Final extension	68°C	5 minutes
Hold	4-10°C

Team:Aix-Marseille/Experiments/Protocols