Click to add water
*: This time needs to be adjusted as different length of the DNA sequence and according to different enzymes. For enzyme pfu, 60s is for DNA sequence of length 2000bp. For enzyme taq, 60s is for DNA sequence of length 1000bp. The minimum time is 30s in this column.
*1: The template here is the bacterial colony so volume is 0.
*2: This time needs to be adjusted as different length of the DNA sequence and according to different enzymes. For enzyme pfu, 60s is for DNA sequence of length 2000bp. For enzyme taq, 60s is for DNA sequence of length 1000bp. The minimum time is 30s in this column.
300ul 50% glycerin + 300ul bacteria, preserve at temperature of -80ºC.
Golden gate assembly:
*: The volume of template is decided by the number of samples needed to be added, normally each sample needs 1ul, further calculations are needed for samples with large difference in length of DNA sequences.
Select one monoclonal bacteria
Add 5ul proper antibiotics into 5ml of LB cultivation medium
Add this bacteria into the solution
Extraction of pilin in vivo:
1. Scrape the bacteria from 20 petri dishes, for each one use 1ml of 1x M9 culture medium (500ul to scrape and 500ul to wash)
2. Collect 20ml into a centrifuge tube, centrifuge for 4000rpm, 4ºC and 15min, precipitate the bacteria.
3. Dump the supernatant and wash tube by 30ml of 150mM ethanolamine(pH=10.5), resuspended the bacteria in this solution.
4. Pour the solution into a waring blender, use 20ml ethanolamine to wash the centrifuge tube for three times, and gain a solution for total 90ml in the blender.
5. Whip the solution using low speed for 2min.
6. Pour the solution into new centrifuge tubes, use 10ml of ethanolamine to wash the blender and pour into the tubes.
7. Centrifuge the product using 5000g, 4ºC for 30min and collect the supernatant.
8. Add TritonX100 detergent to rich a final concentration of 6mM
9. Put the mixture on a shaker for 100rpm, 45min to dissolve the Triton.
10. Add ethanolamine to the solution to lower the concentration of Triton to 2mM
11. Pour the mixture into a stirring filtration unit that had a 100kDa molecular weight cutoff membrane filter made from polyethersulfone(Omega membrane 100K 76mm, Pall Corporation).
12. The sample was filtered under nitrogen gas of 69kPa.
13. The sample on the filter is collected and then washed with 100ml of water for four times.
14. Collect the residues and scrap the surface into 500ul of water.
15. Repeat procedure 14 twice to yield a suspension in 1.5ml of water.
Trans5α competent cell transformation
Transformation must be done in the laminar flow hood!!
1. Place the competent cells flat on the ice to dissolve, and divide it into 50 uL's.
2. Add plasmid sample to the competent cells, shake it gently for several times to mix it well.
3. Ice bath for 30 min.
4. Heat shock at 42ºC for 60s.
5. Ice bath for 5 min.
6. Add 300 uL anti-LB medium.
7. Put it into the shaker for 1h at 37°C.
8. 100 uL bacterial solution was applied and coated on the chloramphenicol-resistant plate.
9. Culture it overnight at 37°C.
1. Centrifuge the bacteria to let them precipitate to the bottom, and pour away the supernatant.
2. Add 150 uL P1 and let the bacteria resuspended; then mix them evenly by shaking.
3. Add 150 uL P2 and reverse it slowly for 8 times; mix well to turn the solution purple.
4. Add 350 uL P5, then flip it over for 20 times immediately after addition until the purple color was completely eliminated and DNA precipitate appeared.
5. Centrifugate the solution for 10 min at 12000 rpm to precipitate genomic DNA and proteins.
6. Add 650 uL supernatant to the adsorbent column and place the adsorbent column in the collection tube.
7. Centrifuge for 2 min at 12000 rpm, and pour out the liquid.
8. Add 300 uL ethanol, let stand for 2 min, and centrifuge it for 1 min at 12000rpm.
9. Pour out the liquid, let stand for 2 min, and centrifuge it for 1 min at 12000rpm.
10. Transfer the column to a new PVC tube and let the ethanol evaporate completely at 42°C.
11. Add 50 uL water to the column, centrifuge for 1min at 12000rpm, and elute the plasmid.
1. Column equilibration: Add 500ul equilibrium liquid (BL) to the absorption column CA2. Then centrifuge it at 12,000 revolutions per minute for one minute, and pour out the waste liquid.
2. Cutting the gel: Make sure to cut it precisely and excise the excess part. Then, put it into the centrifugal tube to measure its weight. (Note that if the gel weight 0.1 gram, it has a volume of 100ul.
3. Add PN solution that has equal volume into the gel, and heat up to 50 degrees Celsius to melt it.
4. Add the melted mixture (liquid) to the absorption column, and then wait for 2 minutes. After that, centrifuge the liquid at 12,000 revolutions per minute for one minute, and pour out the liquid.
5. Add 600ul buffer PW, and repeat Step 4. (Remember to wait for 2 minutes before putting it into the centrifuge.)
6. Repeat Step 5.
7. After placing the absorption column back into the collection tube, we centrifuge it at 12,000 revolutions per minute for 2 minutes, which help us to eliminate the buffer PW. Then we heat it up to 55 degrees Celsius to let all the ethanol fully evaporate.
8. Change the collection tube to a clean, unused PVC tube. Add 30ul water into it, and wait for two minutes. After that, we centrifuge it at 12,000 revolutions per minute for one minute.
9. Collect the DNA solution remain in the PVC tube, and discard the absorption column.
Inoculation: Select one colony into 5ml TB medium (1% glycerol kanamycin) for 24 hours at 30 degrees Celsius. Then spread the bacteria solution on K and LB medium for 30 degrees Celsius overnight. After that, scrape the bacteria from the LB medium and resuspend them in 6ml M9 solution. Spread 300ul nano2-BL21 solution on M9 plate with IPTG plus Kanamycin plus 0.5% glycerin for a total of 20. Then cultivate them at 30 degrees Celsius for 48 hours.
1. Assemble the electrophoresis cell with the shorter glass plates placed inward. Fill the inner core with SDS buffer. If there are no leaks, add extracellular fluid to the red seal.
2. Spot the samples. Be aware of the position of marker.
3. Connect the electrophoresis cell to the power supply and perform electrophoresis with a voltage of 200V and a current of 300mA for 70 minutes.
4. Add transfer buffer into gel cassette. Soak nitrocellulose membrane, gel and blotting paper in it. The gel and nitrocellulose membrane are sandwiched by three pieces of blotting paper on each side. Place it in transfer apparatus and fill it with transfer buffer. Run transfer apparatus for 60 minutes on 100V and 300mA.
5. Remove the membrane from the transfer apparatus and place it in a blotting box. Wash it for 5 minutes with 10 ml TBST.
6. Place it in blocking buffer for 40 minutes.
7. Incubate the membrane in diluted primary antibody for an hour.
8. Wash the membrane for three times, 5 minutes each time in TBST.
9. Incubate the membrane in diluted secondary antibody for an hour.
10. Wash the membrane for three times, 5 minutes each time in TBST.
11. Develop blots with substrate solution, reagent A 3ml, reagent B 3ml. Mix and add them to a piece of gel. After 5 to 15 minutes without being exposed to light, check the result. Take a picture of it and store it.
12. Wash off the substrate solution and store it in darkroom.