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− | #Incubate 20 min at 80°C (inactivation of restriction enzyme) | + | #Incubate 20 min at 80°C (inactivation of restriction enzyme) |
===Ligation=== | ===Ligation=== |
Revision as of 09:24, 13 June 2016
Contents
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).
- 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 80mL 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 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 |
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
- 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 |
- Incubate for 45 min at 37°C
- Incubate 20 min at 80°C (inactivation of restriction enzyme)
Ligation
- 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
- {| class="wikitable"
|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 |}
- 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