IGEM:Peking/2007/Count-Procedure-tandem-oriT

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Tandem OriT(F-OriT for example)

Step 0:Preparation of Competent Cells

  1. <Preparation of Competent Cells STANDARD PROTOCOL>
  2. Antibiotic efficiency testing

Step 1: pre-Exp

  1. Get I14032,J23066,J61003(from BioBricks).
  2. Dissolution with 15ul dH20,7；1ul for transformation ,14Ul stored in -20℃.
  3. <Transformation STANDARD PROTOCOL> .
  4. Screen positive colonies from plate ,Culture in liquid LB and mini-prep, stored in -20℃.
  5. synthesize OriT(J01002) primer.

Step 2:PCR J01002

  1. <PCR STANDARD PROTOCOL>,Test with: <Electrophoresis STANDARD PROTOCOL>.
  2. <PCR Purification Product STANDARD PROTOCOL>, Test with:<Electrophoresis STANDARD PROTOCOL>.


step 3:OriT(J01002)-> pSB1A2

  1. Get PCR product J01002 from refrigerator.
  2. <Transformation STANDARD PROTOCOL> using pEASY-T3 as Vector,OriT(J01002) as fragment.(LB/Amp+,IPTG/X-Gal as screening marker).
  3. Screen Positive Colonies from Plate ,Culture in liquid LB for 12hours and mini-prep, Test with <Restriction Enzymes Digestion STANDARD PROTOCOL>(EcoRI/PstI).
  4. J01002for extraction:<Restriction Enzymes Digestion STANDARD PROTOCOL>, <Electrophoresis STANDARD PROTOCOL >, <DNA Gel Extraction STANDARD PROTOCOL>, test gel extraction efficiency with <Electrophoresis STANDARD PROTOCOL>.
  5. Get E0040-pSB1A2 plasmid from refrigerator.
  6. 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>.
  7. <DNA ligation STANDARD PROTOCOL>(J01002 as fragment,pSB1A2 as vector).
  8. <Transformation STANDARD PROTOCOL>
  9. Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
  10. Test with <restriction enzymes Digestion STANDARD PROTOCOL> ，if Positive , Go next step.
  11. Sequencing OriT(J01002)

Step 4:Step 3 <- Key(J23066)

  1. Get step 3 postive plasmid ，J23066　plasmid from refrigerator.
  2. 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>.
  3. 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>.
  4. <DNA ligation STANDARD PROTOCOL>
  5. <Transformation STANDARD PROTOCOL>
  6. Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
  7. Test with <restriction enzymes Digestion STANDARD PROTOCOL> ，if Positive , Go next step.


Step 5:step4 <- OriT(J01002)

  1. Get J01002，Step 4 positive plasmid from refrigerator.
  2. 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>.
  3. 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>.
  4. <DNA ligation STANDARD PROTOCOL>
  5. <Transformation STANDARD PROTOCOL>
  6. Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
  7. 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

  1. Get R0010，Step 5 positive plasmid from refrigerator.
  2. 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>.
  3. 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>.
  4. <DNA ligation STANDARD PROTOCOL>
  5. <Transformation STANDARD PROTOCOL>
  6. Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
  7. Test with <restriction enzymes Digestion STANDARD PROTOCOL> ，if Positive , Go next step.
  8. update Product State on WIKI
  9. Sequencing

Step 7:Step 6 <- E0240

  1. Get E0240，Step 6 positive plasmid from refrigerator
  2. 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>.
  3. 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>.
  4. <DNA ligation STANDARD PROTOCOL>
  5. <Transformation STANDARD PROTOCOL>
  6. Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
  7. Test with <restriction enzymes Digestion STANDARD PROTOCOL> ，if Positive , Go next step.
  8. Update Product State on WIKI
  9. 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>
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