IGEM:Peking/2007/Count-Procedure-Riboregulator

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Step 0: pre-Exp)
Line 12: Line 12:
#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 .
#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 .
#Order the primers from TaKaRa.
#Order the primers from TaKaRa.
-
#1 cycle PCR to synthesize dsDNA.
+
#5 cycle PCR to synthesize dsDNA.
-
#Enzymatic Digestion with the proper enzyme and cloning into vector (pSB1A2).
+
#Optional:Enzymatic Digestion with the proper enzyme and cloning into vector (pSB1A2).
-
#For some taRNAs, assemble them into double taRNAs with Standard Method.;
+
#For some taRNAs, assemble them into double taRNAs with Standard Method (''"2 primer strategy"''').;
===Step 2:E0040 -> pSB3K3===
===Step 2:E0040 -> pSB3K3===
Line 26: Line 26:
#Update Product State on WIKI
#Update Product State on WIKI
-
===Step 3:R0040 <- crRNA===
+
===Step 3:E0040(pSB3K3) <- B0015===
 +
#Get B0015,E0040(pSB3K3) plasmid from refrigerator.
 +
#<Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors E0040(pSB3K3):SpeI/PstI. Fragment B0015:XbaI/PstI (0.5uL for each enzyme)
 +
#<Electrophoresis STANDARD PROTOCOL>,<DNA Gel Extraction 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
 +
 
 +
===Step 4:R0040 <- crRNA===
#Get R0040 plasmid from refrigerator, crRNA from Step1 PCR.
#Get R0040 plasmid from refrigerator, crRNA from Step1 PCR.
#<Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors R0040:SpeI/PstI. Fragment crRNA: XbaI/PstI (0.5uL for each enzyme)
#<Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors R0040:SpeI/PstI. Fragment crRNA: XbaI/PstI (0.5uL for each enzyme)
Line 37: Line 47:
#Sequencing  
#Sequencing  
-
===Step 4:Step3 -> Step2===
+
===Step 5:Step4 -> Step3===
#Get Step3,Step2 positive plasmid from refrigerator.
#Get Step3,Step2 positive plasmid from refrigerator.
-
#<Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors Step2: EcoRI/XbaI,fragment Step3:EcoRI/SpeI  (0.5uL for each enzyme)
+
#<Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors Step3: EcoRI/XbaI,fragment Step4:EcoRI/SpeI  (0.5uL for each enzyme)
#<Electrophoresis STANDARD PROTOCOL>,<DNA Gel Extraction STANDARD PROTOCOL>
#<Electrophoresis STANDARD PROTOCOL>,<DNA Gel Extraction STANDARD PROTOCOL>
#<DNA ligation STANDARD PROTOCOL>
#<DNA ligation STANDARD PROTOCOL>
Line 48: Line 58:
#Sequencing
#Sequencing
-
===Step 5:R0010 <- taRNA ===
+
===Step 6:R0010 <- taRNA ===
#Get R0010,taRNA positive plasmid from refrigerator.
#Get R0010,taRNA positive plasmid from refrigerator.
#<Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors R0010: SpeI/PstI,fragment taRNA:XbaI/PstI (0.5uL for each enzyme)
#<Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors R0010: SpeI/PstI,fragment taRNA:XbaI/PstI (0.5uL for each enzyme)
Line 55: Line 65:
#<Transformation STANDARD PROTOCOL>
#<Transformation STANDARD PROTOCOL>
#Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
#Screen Positive Colonies from Plate ,Culture in liquid LB and mini-prep, stored in -20℃
-
#Step5 competent cell.
 
#Test with <restriction enzymes Digestion STANDARD PROTOCOL> ,if Positive , Go next step.
#Test with <restriction enzymes Digestion STANDARD PROTOCOL> ,if Positive , Go next step.
#update Product State on WIKI
#update Product State on WIKI
#Sequencing
#Sequencing
-
===Step 6: Step4 and Step5 Cotransformation===
+
===Step 7:Step5 <- B0015===
 +
#Get Step5,B0015 plasmid from refrigerator.
 +
#<Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors Step6:SpeI/PstI. Fragment B0015:XbaI/PstI.(0.5uL for each enzyme)
 +
#<Electrophoresis STANDARD PROTOCOL>,<DNA Gel Extraction 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.
 +
#Step6 competent cell.
 +
#Update Product State on WIKI
 +
 
 +
===Step 8: Step5 and Step7 Cotransformation===
#Get step4 positive plasmid and step5 competent cell from refrigerator.
#Get step4 positive plasmid and step5 competent cell from refrigerator.
#transform step4 plasmid into step5 cells. <transformation STANDARD PROTOCOL>  
#transform step4 plasmid into step5 cells. <transformation STANDARD PROTOCOL>  
#Screen Positive Colonies from Plate ,Culture in liquid LB.
#Screen Positive Colonies from Plate ,Culture in liquid LB.
-
===Step 7: Induction and Measure the Fluorescence Intensity===
+
===Step 9: Induction and Measure the Fluorescence Intensity===
#Culture until OD = 0.4 ,diluted to 1:1000.  
#Culture until OD = 0.4 ,diluted to 1:1000.  
#Three Groups are divided;
#Three Groups are divided;

Revision as of 04:39, 31 August 2007

Contents

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 dH2O;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℃

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. <Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors pSB3K3:EcoRI/XbaI. Fragment E0040: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

Step 3:E0040(pSB3K3) <- B0015

  1. Get B0015,E0040(pSB3K3) plasmid from refrigerator.
  2. <Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors E0040(pSB3K3):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. Update Product State on WIKI

Step 4:R0040 <- crRNA

  1. Get R0040 plasmid from refrigerator, crRNA from Step1 PCR.
  2. <Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors R0040:SpeI/PstI. Fragment crRNA: 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 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

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;
    1. Group A Control group, with nothing add. ;
    2. Group B Add IPTG (concentration should be 0.01mmol/L,0.1mmol/L,1mmol/L);
    3. Group C Add IPTG (concentration should be 0.01mmol/L,0.1mmol/L,1mmol/L) and aTc (30ng/mL), induced separately.;
  3. Incubate at 28℃
  4. <Measure the Fluorescence Intensity STANDARD PROTOCOL>
Personal tools