Team:SJTU-BioX-Shanghai/Protocols
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Project introduction. Inspired by the natural regulator of circadian bioclock exhibited in most eukaryotic organisms, our team has designed an E.coli-based genetic network with the toxin-antitoxin system so that the bacterium oscillates between two states of dormancy and activity (more...)
Contents |
Protocols
Standard biobrick preparation
- Centrifuge the distribution plates for a while so that the dry DNA precipitates onto the bottom of the wells;
- Penetrate the aluminum foil with the tip of a pipette (Caution: avoid damaging other wells);
- Add 15μl double-distilled water (ddH2O);
- Take 1~2μl to perform following operations.
Transduction
- Set the water bath to 42℃;
- Mark and place an EP tube of E.coli competent cells (ca.100μl) in a freezing box;
- Add 1~2μl plasmid solution and leave the tube in the freezing box for 30min;
- Heat shock the cells in the water bath for an exact time of 90s;
- Take out the tube and place it in the ice box for another 5min;
- Add 400μl (or 1000μl) of liquid medium into the tube and mark it;
- Shaking cultivate at 37℃ for 0.5~2h; meanwhile prepare the culture plates with corresponding antibiotic in the medium;
- Inoculate and cultivate the plates upside-down in a 37℃ incubator for 12~14h.
E.coli culture
- Add antibiotics of 0.1% concentration into a culture tube with 4~5ml liquid medium inside;
- Pick a single colony with a pipette tip from a culture plate and inoculate it into the liquid medium;
- Shaking culture in a 37℃ incubator overnight (ca. 12~16h).
Bacterium strain storage
- Prepare a 1.5ml EP tube and mark it;
- Add 400μl of 80% glycerin into the tube;
- Add 600μl of cultivated liquid medium into the tube;
- Store the tube in a -80C℃ fridge.
Agarose gel electrophoresis
- Gel preparation:
- Ingredients:
- (for a smaller piece of gel) 0.3g of agarose + 30ml of TBE + 4μl of EB;
- (for a larger piece of gel) 0.8g of agarose + 80ml of TBE + 4μl of EB;
- Ingredients:
- Weigh out the required amount of agarose powder onto a piece of weighing paper;
- Add the agarose powder into a glass bottle specific for gel preparation;
- Add the required amount of TBE buffer into the bottle;
- Microwave the liquid with the bottle lid slightly open until it boils, so that the agarose powder gets well dissolved; meanwhile prepare the gel plates and ensure the right comb is fixed;
- Cool the bottle in a sink;
- When the solution feels warm but not hot (ca.60℃), add the required amount of EB with a pipette specific for gel preparation into the bottle;
- Shake the bottle carefully so that the EB is well mixed;
- Pour the liquid onto the gel plate;
- Wait for the liquid to be cured.
- Spotting:
- Place a disposable glove on the experiment table;
- Pipette correct amounts of loading buffer (so that the buffer will be diluted to a correct concentration) onto the glove to form a lane of buffer drops;
- Pipette correct amounts of samples into the buffer drops and mix them well;
- Pipette the correct DNA ladder (or marker) into a gel lane, usually the first or the middle-most one;
- Pipette the mixed samples into lanes of the gel; watch out for possible leakage which occurs when the pipette tip gets too deep and penetrates through the gel lane.
- Electrophoresis:
- Voltage selection:
- (for a general resolution) 160V;
- (for a high resolution) 120V (and correspondingly longer electrophoresis time);
- Voltage selection:
- After spotting the samples, install the lid of electrophoresis chamber and switch the output on;
- When the fastest lane runs to the center (or 2/3 the length if follow-up gel purification is intended) of the gel, switch off the output and transfer the gel into the UVP system;
- Power on UVP and observe the gel; take photos when necessary;
- Switch off the system and discard the gel into a special rubbish bin.
Double digestion
- System selection:
- For identification only, choose the 20μl system;
- For following ligation, choose the 100μl system;
- Refer to the Takara inventory for the correct buffer. Take EcoRI and PstI for an example:
- 20μl system:
- 0.5μl EcoRI
- 0.5μl PstI
- 2μl 1*H
- 10μl plasmid (100ng/μl)
- 7μl ddH2O
- 100μl system:
- Plasmid 30μl (100ng/μl)
- EcoRI 3μl
- PstI 3μl
- 1*H 10μl
- ddH2O 54μl
- 20μl system:
- Prepare the system on a freezing box;
- Place the system under 37℃ temperature for 2~3h (for identification) or 9~12h (for ligation).
Tips for digestion
- Better limit the volume of restriction enzymes to less than 1/10 of the whole system, for the star activity would be enhanced under high concentration conditions. In most cases it is about 1/20 of the whole system.
- To avoid the case in which DNA concentration is too low and hence electrophoresis yields no result, ensure a minimum DNA amount of 50ng.
- For identification only, an estimated amount of 0.5μl of enzyme (8~20U) should correspond to 1μg of DNA; For ligation, 1μl of enzyme to 1μg of DNA.
- For identification only, a digestion time of 2~3h is enough; for ligation it’s better to leave the system overnight, otherwise massive false positive colonies would occur during the following steps of transduction and culture.