IGEM:Peking/2007/Count-Procedure-Riboregulator: Difference between revisions
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#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℃ | ||
#Test with <restriction enzymes Digestion STANDARD PROTOCOL> ,if Positive , Go next step. | #Test with <restriction enzymes Digestion STANDARD PROTOCOL> ,if Positive , Go next step. | ||
===Step 3:taRNA | ===Step 3:taRNA+pSB1A3=== | ||
#Get J23066,pSB1A3 plasmid from refrigerator. | #Get J23066,pSB1A3 plasmid from refrigerator. | ||
#<Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors taRNA:EcoRI/XbaI. Fragment:EcoRI/SpeI (0.5uL for each enzyme) | #<Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors taRNA:EcoRI/XbaI. Fragment:EcoRI/SpeI (0.5uL for each enzyme) | ||
Line 24: | Line 24: | ||
#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℃ | ||
#Test with <restriction enzymes Digestion STANDARD PROTOCOL> ,if Positive , Go next step. | #Test with <restriction enzymes Digestion STANDARD PROTOCOL> ,if Positive , Go next step. | ||
===Step 4:R0010+Step 3=== | ===Step 4:R0010+Step 3=== | ||
#Get R0010,Step 3 positive plasmid from refrigerator. | #Get R0010,Step 3 positive plasmid from refrigerator. |
Revision as of 06:20, 7 July 2007
taRNA&crRNA Testing
Step 0: pre-Exp
- Get PARTS:pSB1A3, pSB3K3, pSB1AK3, R0010, J23066 (from BioBricks)
- Dissolution with 15ul dH20,7;1ul for transformation ,14Ul stored in -20℃.
- <Transformation STANDARD PROTOCOL>
- Screen positive colonies from plate ,Culture in liquid LB and mini-prep, stored in -20℃
Step 1:taRNA & crRNA synthesize
- Synthesize taRNA+crRNA
- Sequencing
Step 2:E0040+pSB3K3
- Get E0040,pSB3K3 plasmid from refrigerator.
- <Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors taRNA:EcoRI/XbaI. Fragment:EcoRI/SpeI (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.
Step 3:taRNA+pSB1A3
- Get J23066,pSB1A3 plasmid from refrigerator.
- <Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors taRNA:EcoRI/XbaI. Fragment:EcoRI/SpeI (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.
Step 4:R0010+Step 3
- Get R0010,Step 3 positive plasmid from refrigerator.
- <Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors :EcoRI/XbaI,fragment:EcoRI/SpeI (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
- Sequencing
Step 5:Step1+Step 2
- Get synthesized RNA,Step 2 positive plasmid from refrigerator.
- <Restriction Enzymes Digestion STANDARD PROTOCOL>,Vectors :EcoRI/XbaI,fragment:EcoRI/SpeI (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
- Sequencing
Step 6: Step 4 and Step 5 Cotransformation
- Get Step 4 positive plasmid,Step 5 positive plasmid from refrigerator.
- <Cotransformation STANDARD PROTOCOL>
- Screen Positive Colonies from Plate ,Culture in liquid LB.
Step 7: Induction and Measure the Fluorescence Intensity
- Culture until OD = 0.4 ,diluted to 1:1000.
- 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;
- incubate at 28℃
- <Measure the Fluorescence Intensity STANDARD PROTOCOL>