Difference between revisions of "Team:Aix-Marseille/Protocols"

Plasmids transformation :
Add 20 ng of plasmid to 100 µL of competent cells thawed in ice
Incubate 30-45 min in ice
Thermal shock : put tubes in the Thermomixer at 42°C during 2 min
Incubate 5 min in ice
Add 900 µL of LB
Incubate 1 hour at 37°C with agitation
Spread 100 µL on LB limp (with antibiotic)
To make negative control, follow the same procedure but without add plasmids and spread 300 µL
Ligation transformation:
Add 20 ng of ligation’s product to 100 µL of competent cells thawed in ice
Incubate 30-45 min in ice
Thermal shock : put tubes in the Thermomixer at 42°C during 2 min
Incubate 5 min in ice
Add 900 µL of LB
Incubate 1 hour at 37°C with agitation
Centrifuge 5 min at 5000 rpm
Eliminate 850 µL of medium
Suspend the pellet
Spread 150 µL on LB limp (with antibiotic)
To make negative control, follow the same procedure but without add plasmids and spread 300 µL

Make a culture of your bacteria in LB medium and let it grow until bacteria are in exponential phase (OD600 = 0.5).
Cells are cold centrifuge 10 min at 3500 rpm.
The pellet is slowly suspended in 80 mL of Tfb1 buffer (300mM KOAc, 0.05M MnCl2, 0.1M KCl, 0.01M
CaCl2, 15% Glycerol (see next section “Preparation of Tbf1 and Tbf2 buffer”).
After another 5 min cold centrifugation at 3500 rpm, the pullet is suspended in 8 mL of Tbf2 Buffer
(0.01mM NaMOPS pH 7, 0.075M CaCl2, 0.01M KCl, 15% Glycerol)
Incubate 15 min in ice. Aliquot 200µL of cell suspension in sterile Eppendorf tubes. The cell
suspension is conserved at -80°C.

For 200 mL of culture:
Preparation of 80 mL of Tbf1 Buffer:

KAc 1M	2.4 mL
MnCl2 0.5M	8 mL
KCl 1 M	8 mL
CaCl2 0.1M	8 mL
Gly 80%	15 mL
H2O	38.6 mL

Preparation of 8 mL of Tbf2 Buffer:

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

50% glycerol	6.25 mL Glycerol 80%
20 mM EDTA	0.4 mL EDTA 0.5 M
0.05% Bromophenol blue	≈0.05g Bromophenol blue
0.05% Xylene cyanol	≈0.05g xylene cyanol
1% SDS	1 mL SDS 10%

DNA	Between 50 and 100 ng
EcoRI-HF	0.2 µL
PstI	0.2 µL
10X NEBuffer 2	2 µL
100X BSA	0.2 µL
H2O	To 20 µL

Incubate all digest reactions at 37°C for 1 hour and then add 3 µL of SES 4X and migrate 30 min at
150V on a 1% agarose gel.

Upstream part :

Upstream part plasmid	500 ng
EcoRI-HF	1 µL
PstI	1 µL
10X NEBuffer 2	2.5 µL
100X BSA	0.5 µL
H2O	To 50 µL

Downstream part :

Downstream part plasmid	500 ng
Xba I	1 µL
Spe I	1 µL
10X NEBuffer 2	2.5 µL
100X BSA	0.5 µL
H2O	To 50 µL

Destination plasmid:

Destination plasmid	500 ng
EcoRI-HF	1 µL
PstI	1 µL
DpnI	1 µL
10X NEBuffer 2	2.5 µL
100X BSA	0.5 µL
H2O	To 50 µL

Incubate the three restriction digest reactions at 37°C for 10 minutes and then heat inactivate at 80°C for 20 minutes.

Upstream part digestion	2 µL
Dowstream part digestion	2 µL
Destination plasmid	2 µL
10X T4 DNA ligase buffer	2 µL
T4 DNA ligase	1 µL
H2O	11 µL

Incubate at RT for 1 hour.

The sequences are already prepared like BioBrick Assembly, they also have the prefixe and suffixe
This protocol is made to prepare the DNA, digest, ligate and transform it.

• 1- Resuspend gBlocks Gene fragment to a final concentration of 10 ng/µL in TE or AE (elution buffer). The tube contain 1000 ng of gBlocks Gene fragment so you have to add 100µL of AE
• 2- Mix well, a vortex shall be used

Digestion E/P

Digest 100 ng in 50µL

Component	50 µL of reaction
DNA	10 µL
Net buffer 2.1	5 µL
H2O	33 µL
EcoRI	1 µL
Pst I	1 µL

1- Incubate for 45 min at 37°C 2- Incubate 20 min at 80°C (inactivation of restriction enzyme)

Component	50 µL of reaction
DNA	10 µL
Net buffer 2 (10X)	5 µL
BSA (100X)	0.5 µL
H2O	33 µL
EcoRI	1 µL
Pst I	1 µL

Ligation

• 1- Ligate 50 ng vector with a 3X molar excess of gBlocks Gene fragments in fresh T4 DNA ligase buffer diluted to 1X and 400 U T4 DNA ligase for a total volume of 20 µL

Component	20 µL of reaction
PSBIC3 (or other vector) at 12.5 ng/µL	2 µL
insert digested (at 37.5 ng/µL)	12 µL
T4 ligase buffer	2 µL
T4 ligase 400U	1 µL
H2O	3 µL

• 1- Incubate 2h at room temperature

Transformation

• 1- Add 10 µL of ligation’s product to 100 µL of competent cells thawed in ice (TGI cells)
• 2- Incubate 45 min in ice
• 3- Thermal shock: put tubes in the thermomixer at 42°C during 2 min
• 4- Incubate 5 min in ice
• 5- Add 900 µL of LB
• 6- Incubate 1 hour at 37°C with agitation
• 7- Centrifuge 5 min at 5000 rpm
• 8- Eliminate 850 µL of supernatant
• 9- Suspend the pellet in the 150µL of remaining medium
• 10- Spread 150 µL on LB limp (with antibiotic)

To make negative control, follow the same procedure but without add plasmids and spread 300 µL

Take the EconoTaq PLUS 2X Master Mix only when all your tubes are ready and keep it on ice all the time you need it.
Add the EconoTaq PLUS 2X Master Mix at the end, when your tubes contain all the other components..

	25 µL Reaction	50 µL Reaction	100 µL Reaction	Final concentration
EconoTaq PLUS 2X Master Mix	12.5 µL	25 µL	50 µL	1X
Forward Primer (100 pmol/µL)	0.5 µl	0.5 µL	1 µL	1 pmol/µL (1µM)
Reverse Primer (100 pmol/µL)	0.5 µL	0.5 µL	1 µL	1 pmol/µL (1µM)
DNA Template*	0.5 µL	1 µL	1 µL
Water nuclease-free	11 µL	23 µL	47 µL

*One colony in 50 µL of Water.15-20 min at 80°C. 5 min at 96°C.

Cycling Step	Temperature	Time	Number of cycles
Initial denaturation	98°C	3 min	1
Denaturation	95°C	10 sec	X 27
Annealing	50-65°C	40 sec	X 27
Extension	72°C	1 min/kb	X 27
Hold	16°C	Indefinitely

Take the Q5 High Fidelity DNA Polymerase only when all your tubes are ready and keep it on ice all the time you need it.
Add the Q5 High Fidelity DNA Polymerase at the end, when your tubes contain all the other.

Component	50 µL reaction
5X Q5 Buffer	10 µL
dNTP (10 µM)	1 µL
Primer Forward 10 µM	2.5 µL
Primer Reverse 10 µM	2.5 µL
DNA	1 colony* or 1 µL
Q5 Polymerase	0.5 µL
Water	To 50 µL

*One colony in 50 µL of Water. 15-20 min at 80°C. 5 min at 96°C.

Cycling Step	Temperature	Time	Number of cycles
Initial denaturation	98°C	10 min if DNA is from a colony else 3 min	1
Denaturation	98°C	10 sec	X 27
Annealing	55°C	20 sec	X 27
Extension	72°C	30 sec/kb	X 27
Hold	16°C	Indefinitely

This is the plasmid purification kit of the Igem-AMU team of 2014.

Use the kit with gloves and without fire.

Cultivate and harvest bacterial cells	Harvest the bacterial cells in a 2 mL tube	Centrifuge 30 sec at 11.000 g
Cell lysis		Add 250µL of Buffer A1 and suspend the cells Then Add 250µL of Buffer A2 Incubate 5 min at RT Add 300 µL of Buffer AE3
Clarification of the lysis	Shake gently until the blue disappear	Centrifuge 5-10 min at 11.000 g
Bind DNA	Load supernatant on a new column with a discard tube below	Centrifuge 1 min at 11.000 g
Wash silica membrane	Discard the content of the discard tube and replace it below the column	(optional: Add 500 µL of Buffer AW and incubate 5 min at RT) Add 600 µL of Buffer A4 Centrifuge 1 min at 11.000 g
Dry silica membrane	discard the content of the discard tube and replace it below the column	Centrifuge 2 min at 11.000 g
Elute DNA	Place a 1.5 mL Eppendorf tube below the column	Add 50 µL of Buffer AE Incubate 1 min at RT Centrifuge 1 min at 11.000g

Nanodrop measurement:

Add 2µL of buffer AE, close the machine and open it again, then clean up and down with paper.

Add 2µL of buffer AE, close and mesure for the blank, then clean up and down with paper.

Add 2µL of your sample and close it. Don’t forget to note your concentration !

Clean your sample with paper, then clean up and down with AE

Don’t put your finger on the paper when you are cleaning, you could contaminate the sample with YOUR DNA !