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Protocols

Protocol #0 : iGEM General Protocol

Obtaining Biobricks
- EITHER on IDT →
  - Order on IDT when the sequences are not too long (chemical synthesis).
  - The received biobricks are lyophilized, they must be resuspended in water (cf protocole).
- OR by PCR →
  - When sequences are too long and costly to order on IDT, or when a sequence must be added to an existing biobrick :
    - Order primers
    - synthesize sequence by PCR (cf protocole Q5)
    - add the suffixes and prefixes during the synthesis « E, X » and « S, P » (E=EcoRI, X=XbaI, S=SpeI, P=PstI) and homologue sequences to plasmids
    - Do a SLIC (cf protocole) to insert the wanted sequence in the wanted plasmid
    - the Biobrick is obtained
Transform the Bacteria with the Biobrick
1. E/S Digestion of the Biobrick from the original plasmid and reinsertion in the E/S pre-restricted wanted plasmid.
2. Transform the pre-cultivated competent cells, via a thermal shock (DH5-α, TGI, BL21).
3. Add an LB-agar in a petri dish and cultivate the bacteria in rich medium added with antibiotics of interest.
Test the Transformation
1. Run the obtained bacteria culture DNA in a PCR and an electrophoresis gel (qualitative assessment)
2. Purify plasmids (by miniprep Kit) from the previously obtained positive colonies.
3. Do an E/P digestion verification to see if the inserted biobricks have the correct size (On only 100ng of plasmids)
Assemble two biobricks
- “Digestion protocol BioBrick Assembly Kit” et “Ligation protocol BioBrick Assembly”
1. Do an E/S digestion on the first biobrick and an X/P on the second
2. Ligate the two biobricks then insert them into the plasmid
Transforming Bacteria with a new BioBrick
1. E/P Restriction of the biobrick and ligation into a E/P restricted plasmid
2. Transform the pre-cultivated competent cells, via a thermal shock (DH5-α, TGI ou BL21).
3. Pour an LB gel in a petri dish and cultivate the bacteria in an antibiotic rich medium
Test the Transformation
1. Run the obtained bacteria culture DNA in a PCR and pour an electrophoresis gel (qualitative assessment)
2. Purify plasmids (by miniprep Kit) from the previously obtained positive colonies.
3. Do an E/P digestion verification to see if the inserted biobricks have the correct size (On only 100ng of plasmids)
4. Send to sequencing
Repeat steps 4, 5, 6 until obtaining the wanted sequence

Protocol #1 : Preparation of competent bacteria cells

Note : Everything should be done in sterile conditions and on ice (4°C). For 100ml of competent cells

Grow a culture of bacteria in LB medium until it reaches OD600 = 0.5.
- While it grows, prepare Tbf1 and Tbf2 buffers.
Centrifuge cells for 10 minutes at 3500g at 4°C
Resuspend the pellet slowly in 50mL of Tfb1 buffer.
Centrifuge 5 min at 3500g at 4°C.
Resuspend the pellet in 8 mL of Tbf2 buffer.
Incubate 15 min in ice.
Aliquot 200µL of cell suspension in sterile eppendorf tubes and conserve it at -80°C.

Protocol #1bis : Preparation of Tbf1 and Tbf2 buffers

For 100 mL of culture:

Preparation of 50 mL of Tbf1 Buffer

KAc 1M	1.5 mL
MnCl2 0.5M	5 mL
KCl 1 M	5 mL
CaCl2 0.1M	5 mL
Gly 80%	0.93 mL
H2O	32.56 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

Protocol #2 : Transformations

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 for 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)

Ligation transformation

Add 20 ng of ligation 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
Spread 150 μL on LB limp (with antibiotic)

To make negative control, follow the same procedure but without adding plasmids and spreading 300 μL

Protocol #3 : Cloning protocol for IDT sequences

Resuspension

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 Mix well, a vortex shall be used

Digestion E/P

1. Digest 100 ng in 50µL

component	50 µL of reaction
DNA	10 µL
Buffer 2 (10X)	5 µL
BSA (100X)	0.5 µL
H2O	33 µL
EcoRI	1 µL
PstI	1 µL

2. Incubate for 45 min at 37°C 3. Incubate 20 min at 80°C (inactivation of restriction enzyme)

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 Buffer	2 µL
T4 ligase 400 U	1 µL
H2O	3 µL

2. Incubate 2h at room temperature

Transformation

Add 10 µL of ligation’s product to 100 µL of competent cells thawed in ice (TGI cells)
Incubate 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 supernatant
Suspend the pellet in the 150µL of remaining medium
Spread 150 µL on LB limp (with antibiotic)

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

Protocol #4 : Digestion and ligation for verification and BioBrick Assembly

Digestion (verification) protocol

DNA	Between 50 and 100 ng
EcoRI-HF	0.2 µL
PstI	0.2 µL
10X NEBuffer 2	2 µL
H2O	QS 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.

Digestion protocol BioBrick Assembly Kit

Upstream part :

Upstream part plasmid	500 ng
EcoRI-HF	1 µL
SpeI	1 µL
10X NEBuffer 2	5 µL
H2O	To 50 µL

Downstream part, optional, for 3A strategy

Downstream part plasmid	500 ng
XbaI	1 µL
PstI	1 µL
10X NEBuffer 2	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	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.

Ligation protocol BioBrick Assembly

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

Incubate at RT for 1 hour.

Team:Aix-Marseille/Experiments/Protocols

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