Team:Gifu/Protocol
From 2014.igem.org
Protocols
- Restriction Digests
- Ligation
- Transformation
- Densitometry(Nanodrop)
- Colony PCR
- Electrophoresis
- Miniprep
- SDS-PAGE
Restriction Digests
Materials
- Ice and bucket/container
- DNA to be digested
- Buffer M (10x)
- Restriction Enzymes: EcoRI, SpeI, XbaI, PstI
- Incubator
Procedure(an example)
- Add 47ul of DNA to be digested into a 1.5ml microcentrifuge tube.
- Add 5.0ul of10x buffer M.
- Add 1.0ul of EcoRI.
- Add 1.0ul of SpeI.
- There should be a total volume of 50ul. Mix well and spin down briefly.
- Incubate the restriction digest at 38C for 30min. We incubate in an incubator.
- Run a portion of the digest on a gel (6ul), to check that part length is accurate.
Ligation
- Add digested fragment A.
- Add digested fragment B.
- Add ligation mixture.
- Ligate 16C/30 min.
Note: Make the amount of fragment A and B equimolar. And make sure that the volume of ligation mixture is equivalent to the total amount of the volume of fragment A and B. we used a constant temperature water bath.
Transformation
- Deal 25ul of competent cells to each 1.5ml tubes.
- Add 1ul of each solution made by ligation to each tube.
- Close tubes and incubate the cells for 30 min on ice.
- Heat shock the cells by immersion in a pre-heated water bath at 42C for 60 seconds.
- Incubate the cells on ice for 2 minutes.
- Add 225ul of SOC media to each tube.
- Incubate cells at 37C for 30 minutes.
- Inoculate with each solution of cells to each plate with appropriate antibiotics.
- Culture the cells.
Densitometry(Nanodrop,In case of nucleic acid)
- Activate the device.
- Choose “Nucleic Acid” at main menu.
- Wash a space of a sample with distilled water and then wipe gently the top and bottom of the arm.
- Choose a sample type. There are DNA-50(double-strand), DNA-33(single-strand), RNA.
- Pour distilled water to test a blank and after that wipe the top and bottom of the arm.
- Pour the sample to measure the concentration.
Colony PCR
- Collect a colony from a plate and then suspend it in 100 µL of LB medium (liquid).
- Mix the following reagents.
sterile water 27.5 µL 10×PCR buffer 5 µL 2 mM dNTP 5 µL 5 pmol/µL Fw primer 2.5 µL 5 pmol/µL Rv primer 2.5 µL 0.5U/µL Taq polymerase 2.5 µL total 45 µL - Add 5 µL of the suspension to the mixture of reagents.
- Do PCR amplification.
- Measure the length of the DNA.
Electrophoresis (preparing 200 mL of agarose solution)
- Meter 4 g of agarose.
- Add 200 mL of 1× buffer into it.
- Wrap the neck of flask and then make a hole on the wrap.
- Dissolve the agarose with a microwave oven.
- Cool it to a suitable temperature.
- Pour the agarose solution into a mold with a comb.
- Remove bubbles.
- After the gel going solid, dislodge the comb carefully.
- Transfer the mold into a phoresis tank.
- Immerse the mold in 1× buffer.
- Mix 5 µL of DNA solution and 1 µL of 3× dye.
- Pour the mixture into the well.
- Electrophorese at 100V.
- Stop the electrophoresis when the band of dye go up to 3/4 of the gel.
- Pick out the gel and then stain it with ethidium bromide (0.5 µg/mL) for 20 minutes.
- Observe DNA bands with UV transilluminator.
Miniprep
SDS-PAGE
Protein extraction and Sample preparation
- Add 50 µL of culture fluid that was cultured overnight to a liquid culture medium, and then cultivate it for a few hours.
- When the bacteria are not too many, add a proper quantity of IPTG to the liquid culture medium.
- Cultivate it for a few hours and then store it at low temperature.
- Pour 1 mL of the liquid culture into a 1.5ml tube. Centrifuge it(13,000 rpm 4°C 15 minutes) and then discard supernatant fluid. Do twice this step.
- Add 300 µL of PBS to the deposition and then stir it.
- Sonicate the cell suspension with 4 short bursts of 15 sec followed by intervals of 60 sec for cooling.
- Centrifuge(13,000rpm, 4°C, 20minutes)and then collect supernatant into another tube.
- Add 100 ul of PBS to the deposition. Shake it with a vortex and then collect it.
- Pour 8 µL of the supernatant(=cell extract) into a tube, and 8 µL of the precipitation suspension into another tube.
- Add 2 µL of 5×loading dye to the tube and then denature it at 90°C with a block incubator.
- Centrifuge and adjust the sample.
- Apply 10µL of the sample to SDS gel.
Making a SDS-PAGE gel and Electrophpresis
- Mix and shake quickly reagents to adjust the concentration of separation gel.
- Construct a plate for electrophoresis.
- Pour separation gel into a gap of the plate (until about 2 cm below a comb). Note: Wipe a plate for electrophoresis with 70% ethanol. Hold a plate for electrophoresis not to spill the gel.
- Pour a proper quantity of Milli Q water into a gap of the plate and then incubate for an hour.
- Mix and shake quickly reagents for stacking gel (except APS and TEMED).
- Slant the gel plate and absorb multistoried Milli Q water.
- Add APS and TEMED to mixed reagents for stacking gel (step5).
- Fill the gap of the plate with stacking gel and then insert the comb into the gap of the plate. Note: Be careful not to mix bubbles in gel.
- Take out the plate and gel together after stacking gel coagulates.
- Put the plate and gel into a migration tank with the plate toward outside.
- Pour 300 µL of electrophoresis buffer into a phoresis tank. Immerse the gel completely.
- Apply 10 µL of the sample and 5 µL of a marker.
- Electrophorese at 40 mA in the stacking gel and then at 60mA in the separation gel.
- Electrophorese until a pigment comes at an appropriate position.
- Stop electrophoresis and then carefully take the gel. Note: Use tweezers.
- Discard the buffer for electrophoresis and then dye the separation gel with CBB.
- Wash the gel plate and the electrophoretic tank with neutral detergent and then rinse it steadily.
Staining with CBB
- Put the gel into fixing solution.
- Leave the gel to stand with shaking until a band is dyed yellow.
- Collect the fixing solution and then put the gel into CBB dyeing liquid.
- Wrap it and then heat it until it is just before boiling with a microwave oven.
- Remove the wrap carefully and then let vapor out slowly.
- Collect the CBB dyeing liquid and then pour deionized water into a container carrying the gel. Put Kim wipe into the container.
- Infiltrate deionized water into the gel for several tens of minutes. Transfer waste liquid into a tank.
- Take a picture under UV light and then dry the gel and store it.