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Contents 1 Protocols 1.1 Standard biobrick preparation 1.2 Transduction 1.3 E.coli culture 1.4 Bacterium strain storage 1.5 Agarose gel electrophoresis 1.6 Double digestion 1.7 Tips for digestion

Protocols

Standard biobrick preparation

1. Centrifuge the distribution plates for a while so that the dry DNA precipitates onto the bottom of the wells;
2. Penetrate the aluminum foil with the tip of a pipette (Caution: avoid damaging other wells);
3. Add 15μl double-distilled water (ddH2O);
4. Take 1~2μl to perform following operations.

Transduction

1. Set the water bath to 42℃;
2. Mark and place an EP tube of E.coli competent cells (ca.100μl) in a freezing box;
3. Add 1~2μl plasmid solution and leave the tube in the freezing box for 30min;
4. Heat shock the cells in the water bath for an exact time of 90s;
5. Take out the tube and place it in the ice box for another 5min;
6. Add 400μl (or 1000μl) of liquid medium into the tube and mark it;
7. Shaking cultivate at 37℃ for 0.5~2h; meanwhile prepare the culture plates with corresponding antibiotic in the medium;
8. Inoculate and cultivate the plates upside-down in a 37℃ incubator for 12~14h.

E.coli culture

1. Add antibiotics of 0.1% concentration into a culture tube with 4~5ml liquid medium inside;
2. Pick a single colony with a pipette tip from a culture plate and inoculate it into the liquid medium;
3. Shaking culture in a 37℃ incubator overnight (ca. 12~16h).

Bacterium strain storage

1. Prepare a 1.5ml EP tube and mark it;
2. Add 400μl of 80% glycerin into the tube;
3. Add 600μl of cultivated liquid medium into the tube;
4. 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;

1. Weigh out the required amount of agarose powder onto a piece of weighing paper;
2. Add the agarose powder into a glass bottle specific for gel preparation;
3. Add the required amount of TBE buffer into the bottle;
4. 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;
5. Cool the bottle in a sink;
6. 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;
7. Shake the bottle carefully so that the EB is well mixed;
8. Pour the liquid onto the gel plate;
9. Wait for the liquid to be cured.

• Spotting:

1. Place a disposable glove on the experiment table;
2. 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;
3. Pipette correct amounts of samples into the buffer drops and mix them well;
4. Pipette the correct DNA ladder (or marker) into a gel lane, usually the first or the middle-most one;
5. 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);

1. After spotting the samples, install the lid of electrophoresis chamber and switch the output on;
2. 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;
3. Power on UVP and observe the gel; take photos when necessary;
4. 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

1. Prepare the system on a freezing box;
2. Place the system under 37℃ temperature for 2~3h (for identification) or 9~12h (for ligation).

Tips for digestion

1. 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.
2. To avoid the case in which DNA concentration is too low and hence electrophoresis yields no result, ensure a minimum DNA amount of 50ng.
3. 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.
4. 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.