IGEM:Peking/2007/Count-Procedure-Riboregulator

taRNA&crRNA Testing
Important Note:In assembly step titles, parts names are positioned as in the product vectors (5' part on the left). The A->B and B<-A means inserting A into B, indicating A is the fragment and B is the vector. Thus when using ->, the digestion enzymes will be EcoRI/XbaI for the vector and EcoRI/SpeI for the fragment; when using <-, the digestion enzymes will be SpeI/PstI for the vector and XbaI/PstI for the fragment. Refer to the standard assembly method for explanation.

Step 0: pre-Exp

 * 1) Get PARTS：pSB1A2, pSB3K3, pSB1AK3, R0010, R0040(from BioBricks)
 * 2) Dissolution with 15ul d$$H_2O$$；1ul for transformation ,14ul stored in -20℃.
 * 3) 
 * 4) Screen positive colonies from plate ,Culture in liquid LB and mini-prep, stored in -20℃

Step 1:taRNA & crRNA synthesize

 * 1) Synthesize taRNA and crRNA (TaKaRa): Parts with BioBrick prefix and suffix flanked. Designing Primers: two primers covering 5' and 3' of the desired fragment respectively.
 * 2) Order the primers from TaKaRa.
 * 3) 5 cycle PCR to synthesize dsDNA.
 * 4) Optional:Enzymatic Digestion with the proper enzyme and cloning into vector (pSB1A2).
 * 5) For some taRNAs, assemble them into double taRNAs with Standard Method ("2 primer strategy"').;

Step 2:E0040 -> pSB3K3

 * 1) Get E0040，pSB3K3 plasmid from refrigerator.
 * 2) ，Vectors pSB3K3：EcoRI/XbaI. Fragment E0040：EcoRI/SpeI (0.5uL for each enzyme)
 * 3) ,
 * 4) 
 * 5) 
 * 6) Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
 * 7) Test with  ，if Positive, Go next step.
 * 8) Update Product State on WIKI

Step 3:E0040(pSB3K3) <- B0015

 * 1) Get B0015，E0040(pSB3K3) plasmid from refrigerator.
 * 2) ，Vectors E0040(pSB3K3)：SpeI/PstI. Fragment B0015：XbaI/PstI (0.5uL for each enzyme)
 * 3) ,
 * 4) 
 * 5) 
 * 6) Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
 * 7) Test with  ，if Positive, Go next step.
 * 8) Update Product State on WIKI

'''*Note: Step 2,3 are only needed in the construction of the first lock-efficiency-test plasmid. The product of Step 2,3 can be stored and remains useful in the following constructions of the other 2 lock-efficiency-test plasmids.'''

Step 4:R0040 <- crRNA

 * 1) Get R0040 plasmid from refrigerator, crRNA from Step1 PCR.
 * 2) ，Vectors R0040：SpeI/PstI. Fragment crRNA: XbaI/PstI (0.5uL for each enzyme)
 * 3) ,
 * 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 5:Step4 -> Step3

 * 1) Get Step3，Step2 positive plasmid from refrigerator.
 * 2) <Restriction Enzymes Digestion STANDARD PROTOCOL>，Vectors Step3: EcoRI/XbaI，fragment Step4：EcoRI/SpeI (0.5uL for each enzyme)
 * 3) <Electrophoresis STANDARD PROTOCOL>,<DNA Gel Extraction 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 6:R0010 <- taRNA

 * 1) Get R0010，taRNA positive plasmid from refrigerator.
 * 2) <Restriction Enzymes Digestion STANDARD PROTOCOL>，Vectors R0010: SpeI/PstI，fragment taRNA：XbaI/PstI (0.5uL for each enzyme)
 * 3) <Electrophoresis STANDARD PROTOCOL>,<DNA Gel Extraction 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:Step5 <- B0015

 * 1) Get Step5，B0015 plasmid from refrigerator.
 * 2) <Restriction Enzymes Digestion STANDARD PROTOCOL>，Vectors Step6：SpeI/PstI. Fragment B0015：XbaI/PstI.(0.5uL for each enzyme)
 * 3) <Electrophoresis STANDARD PROTOCOL>,<DNA Gel Extraction 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) Step6 competent cell.
 * 9) Update Product State on WIKI

*Note: Step 2,3, Step 4 and Step 6,7 can be parallel.

Step 8: Step5 and Step7 Cotransformation

 * 1) Get step4 positive plasmid and step5 competent cell from refrigerator.
 * 2) transform step4 plasmid into step5 cells. <transformation STANDARD PROTOCOL>
 * 3) Screen Positive Colonies from Plate ,Culture in liquid LB.

Step 9: Induction and Measure the Fluorescence Intensity

 * 1) Culture until OD = 0.4 ,diluted to 1:1000.
 * 2) Three Groups are divided;
 * 3) Group A Control group, with nothing add. ;
 * 4) Group B Add IPTG (concentration should be 0.01mmol/L,0.1mmol/L,1mmol/L);
 * 5) Group C Add IPTG (concentration should be 0.01mmol/L,0.1mmol/L,1mmol/L) and aTc (30ng/mL), induced separately.;
 * 6) Incubate at 28℃
 * 7) <Measure the Fluorescence Intensity STANDARD PROTOCOL>