IGEM:Peking/2007/Count-Procedure-Riboregulator

taRNA&crRNA Testing

Step 0: pre-Exp

1. Get PARTS：pSB1A3, pSB3K3, pSB1AK3, R0010, J23066 (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℃

Step 1:taRNA & crRNA synthesize

1. Synthesize taRNA+crRNA (TaKaRa)
2. Sequencing(TaKaRa)

Step 2:E0040+pSB3K3

1. Get E0040，pSB3K3 plasmid from refrigerator.
2. <Restriction Enzymes Digestion STANDARD PROTOCOL>，Vectors taRNA：EcoRI/XbaI. Fragment：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.

Step 3:taRNA+pSB1A3

1. Get J23066，pSB1A3 plasmid from refrigerator.
2. <Restriction Enzymes Digestion STANDARD PROTOCOL>，Vectors taRNA：EcoRI/XbaI. Fragment: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.

Step 4:R0010+Step 3

1. Get R0010，Step 3 positive plasmid from refrigerator.
2. <Restriction Enzymes Digestion STANDARD PROTOCOL>，Vectors :EcoRI/XbaI，fragment：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 5:Step1+Step 2

1. Get synthesized RNA，Step 2 positive plasmid from refrigerator.
2. <Restriction Enzymes Digestion STANDARD PROTOCOL>，Vectors :EcoRI/XbaI，fragment：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: Step 4 and Step 5 Cotransformation

1. Get Step 4 positive plasmid，Step 5 positive plasmid from refrigerator.
2. <Cotransformation STANDARD PROTOCOL>
3. Screen Positive Colonies from Plate ,Culture in liquid LB.

Step 7: Induction and Measure the Fluorescence Intensity

1. Culture until OD = 0.4 ,diluted to 1:1000.
2. Three Groups are divided;

Group A Control group, with nothing add. ;

Group B Add IPTG (concentration should be 0.01mmol/L,0.1mmol/L,1mmol/L);

Group C Add IPTG (concentration should be 0.01mmol/L,0.1mmol/L,1mmol/L) and aTc（30ng/mL）,induced separately;

1. incubate at 28℃
2. <Measure the Fluorescence Intensity STANDARD PROTOCOL>