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== Protocol #1bis : Preparation of Tbf1 and Tbf2 buffers == | == Protocol #1bis : Preparation of Tbf1 and Tbf2 buffers == | ||
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===Preparation of 50 mL of Tbf1 Buffer=== | ===Preparation of 50 mL of Tbf1 Buffer=== |
Revision as of 13:59, 13 June 2016
Contents
- 1 Protocols
- 1.1 Protocol #0 : iGEM General Protocol
- 1.2 Protocol #1 : Preparation of competent bacteria cells
- 1.3 Protocol #1bis : Preparation of Tbf1 and Tbf2 buffers
- 1.4 Protocol #2 : Transformations
- 1.5 Protocol #3 : Cloning protocol for IDT sequences
- 1.6 Protocol #4 : Digestion and ligation for verification and BioBrick Assembly
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
- When sequences are too long and costly to order on IDT, or when a sequence must be added to an existing biobrick :
- EITHER on IDT →
- Transform the Bacteria with the Biobrick
- E/S Digestion of the Biobrick from the original plasmid and reinsertion in the E/S pre-restricted wanted plasmid.
- Transform the pre-cultivated competent cells, via a thermal shock (DH5-α, TGI, BL21).
- Add an LB-agar in a petri dish and cultivate the bacteria in rich medium added with antibiotics of interest.
- Test the Transformation
- Run the obtained bacteria culture DNA in a PCR and an electrophoresis gel (qualitative assessment)
- Purify plasmids (by miniprep Kit) from the previously obtained positive colonies.
- 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”
- Do an E/S digestion on the first biobrick and an X/P on the second
- Ligate the two biobricks then insert them into the plasmid
- Transforming Bacteria with a new BioBrick
- E/P Restriction of the biobrick and ligation into a E/P restricted plasmid
- Transform the pre-cultivated competent cells, via a thermal shock (DH5-α, TGI ou BL21).
- Pour an LB gel in a petri dish and cultivate the bacteria in an antibiotic rich medium
- Test the Transformation
- Run the obtained bacteria culture DNA in a PCR and pour an electrophoresis gel (qualitative assessment)
- Purify plasmids (by miniprep Kit) from the previously obtained positive colonies.
- Do an E/P digestion verification to see if the inserted biobricks have the correct size (On only 100ng of plasmids)
- 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 |
100X BSA | 0.2 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 |
100X BSA | 0.5 µL |
H2O | To 50 µL |
Downstream part :
Downstream part plasmid | 500 ng |
XbaI | 1 µL |
PstI | 1 µL |
10X NEBuffer 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 | 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.