IGEM:Peking/2007/Count-Procedure-tandem-oriT
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Tandem OriT(F-OriT for example)
Step 0:Preparation of Competent Cells
- <Preparation of Competent Cells STANDARD PROTOCOL>
- Antibiotic efficiency testing
Step 1: pre-Exp
- Get I14032,J23066,J61003(from BioBricks).
- Dissolution with 15ul dH20,7;1ul for transformation ,14Ul stored in -20℃.
- <Transformation STANDARD PROTOCOL> .
- Screen positive colonies from plate ,Culture in liquid LB and mini-prep, stored in -20℃.
- synthesize OriT(J01002) primer.
Step 2:PCR J01002
- <PCR STANDARD PROTOCOL>,Test with: <Electrophoresis STANDARD PROTOCOL>.
- <PCR Purification Product STANDARD PROTOCOL>, Test with:<Electrophoresis STANDARD PROTOCOL>.
step 3:OriT(J01002)-> pSB1A2
- Get PCR product J01002 from refrigerator.
- <Transformation STANDARD PROTOCOL> using pEASY-T3 as Vector,OriT(J01002) as fragment.(LB/Amp+,IPTG/X-Gal as screening marker).
- Screen Positive Colonies from Plate ,Culture in liquid LB for 12hours and mini-prep, Test with <Restriction Enzymes Digestion STANDARD PROTOCOL>(EcoRI/PstI).
- J01002for extraction:<Restriction Enzymes Digestion STANDARD PROTOCOL>, <Electrophoresis STANDARD PROTOCOL >, <DNA Gel Extraction STANDARD PROTOCOL>, test gel extraction efficiency with <Electrophoresis STANDARD PROTOCOL>.
- Get E0040-pSB1A2 plasmid from refrigerator.
- E0040-pSB1A2(for extraction):<Restriction Enzymes Digestion STANDARD PROTOCOL>(EcoRI/PstI), <Electrophoresis STANDARD PROTOCOL> for extraction, <DNA Gel Extraction STANDARD PROTOCOL>, test gel extraction efficiency with <Electrophoresis STANDARD PROTOCOL>.
- <DNA ligation STANDARD PROTOCOL>(J01002 as fragment,pSB1A2 as vector).
- <Transformation STANDARD PROTOCOL>
- Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
- Test with <restriction enzymes Digestion STANDARD PROTOCOL> ,if Positive , Go next step.
- Sequencing OriT(J01002)
Step 4:Step 3 <- Key(J23066)
- Get step 3 postive plasmid ,J23066 plasmid from refrigerator.
- J23066(for extraction):<Restriction Enzymes Digestion STANDARD PROTOCOL>(XbaI/PstI), <Electrophoresis STANDARD PROTOCOL> , <DNA Gel Extraction STANDARD PROTOCOL>,test extraction with efficiency with <Electrophoresis STANDARD PROTOCOL>.
- J01002-pSB1A2(for extraction):<Restriction Enzymes Digestion STANDARD PROTOCOL>(SpeI/PstI), <Electrophoresis STANDARD PROTOCOL> , <DNA Gel Extraction STANDARD PROTOCOL>,test extraction with efficiency with <Electrophoresis STANDARD PROTOCOL>.
- <DNA ligation STANDARD PROTOCOL>
- <Transformation STANDARD PROTOCOL>
- Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
- Test with <restriction enzymes Digestion STANDARD PROTOCOL> ,if Positive , Go next step.
Step 5:step4 <- OriT(J01002)
- Get J01002,Step 4 positive plasmid from refrigerator.
- J01002(for extraction): <Restriction Enzymes Digestion STANDARD PROTOCOL>(XbaI/PstI), <Electrophoresis STANDARD PROTOCOL> , <DNA Gel Extraction STANDARD PROTOCOL>,test extraction with efficiency with <Electrophoresis STANDARD PROTOCOL>.
- J01002-J23066-pSB1A2(for extraction):<Restriction Enzymes Digestion STANDARD PROTOCOL>(SpeI/PstI), <Electrophoresis STANDARD PROTOCOL> , <DNA Gel Extraction STANDARD PROTOCOL>,test extraction with efficiency with <Electrophoresis STANDARD PROTOCOL>.
- <DNA ligation STANDARD PROTOCOL>
- <Transformation STANDARD PROTOCOL>
- Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
- Test with <restriction enzymes Digestion STANDARD PROTOCOL> ,if Positive , Go next step.
- After sequencing, this parts will be named as: Tandem OriT F(I714051)
Step 6:Promoter(R0010) -> Step5
- Get R0010,Step 5 positive plasmid from refrigerator.
- R0010(for extraction): <Restriction Enzymes Digestion STANDARD PROTOCOL>(EcoRI/SpeI), <Electrophoresis STANDARD PROTOCOL> , <DNA Gel Extraction STANDARD PROTOCOL>,test extraction with efficiency with <Electrophoresis STANDARD PROTOCOL>.
- Tandem OriT F(I714051)(for extraction):<Restriction Enzymes Digestion STANDARD PROTOCOL>(EcoRI/XbaI), <Electrophoresis STANDARD PROTOCOL> , <DNA Gel Extraction STANDARD PROTOCOL>,test extraction with efficiency with <Electrophoresis STANDARD PROTOCOL>.
- <DNA ligation STANDARD PROTOCOL>
- <Transformation STANDARD PROTOCOL>
- Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
- Test with <restriction enzymes Digestion STANDARD PROTOCOL> ,if Positive , Go next step.
- update Product State on WIKI
- Sequencing
Step 7:Step 6 <- E0240
- Get E0240,Step 6 positive plasmid from refrigerator
- E0240(for extraction): <Restriction Enzymes Digestion STANDARD PROTOCOL>(XbaI/PstI), <Electrophoresis STANDARD PROTOCOL> , <DNA Gel Extraction STANDARD PROTOCOL>,test extraction with efficiency with <Electrophoresis STANDARD PROTOCOL>.
- R0010-I714051 (for extraction):<Restriction Enzymes Digestion STANDARD PROTOCOL>(SpeI/PstI), <Electrophoresis STANDARD PROTOCOL> , <DNA Gel Extraction STANDARD PROTOCOL>,test extraction with efficiency with <Electrophoresis STANDARD PROTOCOL>.
- <DNA ligation STANDARD PROTOCOL>
- <Transformation STANDARD PROTOCOL>
- Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
- Test with <restriction enzymes Digestion STANDARD PROTOCOL> ,if Positive , Go next step.
- Update Product State on WIKI
- Sequencing
Step 8: Testing
- make F+ Competent Cell.
- transformation:F+ <- OriT
- transformation:F+ <- PlacI-OriT-Key-OriT-E0240
Conjugation Test(efficiency test):
- donor: F+/OriT-pSB1A2
- recipient: DH5α_pSB3K3(or any other plasmid without Amp+)
- Control:donnor: F+, DH5/OriT
- <Conjugation Standard Protocol>
Conjugation Test(Orit Test):
- donor: F+/PlacI-OriT-Key-OriT-E0240
- recipient: DH5α_pSB3K3(or any other plasmid without Amp+)
- Control:donnor: F+, DH5α/PlacI-OriT-Key-OriT-E0240
- <Conjugation Standard Protocol>