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

Step 0:Preparation of Competent Cells

 * 1) 
 * 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) .
 * 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) ,Test with: .
 * 2) , Test with:.

step 3:OriT(J01002)-> pSB1A2

 * 1) Get PCR product J01002 from refrigerator.
 * 2)  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 (EcoRI/PstI).
 * 4) J01002for extraction:, , , test gel extraction efficiency with .
 * 5) Get E0040-pSB1A2 plasmid from refrigerator.
 * 6) E0040-pSB1A2(for extraction):(EcoRI/PstI),  for extraction, , test gel extraction efficiency with .
 * 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>