Team:XHD-Wuhan-A-China/Experiments
Protocols and Experiments
Bacterial genome extration
The experimental steps as follow:
1) The bacteria were cultured at the optimum growth temperature (4 mL) to the logarithmic stage, centrifuged at 12000 rpm for 1 min, and the supernatant was discarded.
2) Add 450 uL sterile water for re-suspension of bacteria solution.
3) Add 50 uL 10% SDS + 5 uL protease K (20 mg/mL) and incubate at 58℃for 2 h.
4) Add 50 uL 5 M NaCl (the final concentration is about 0.5 M).
5) 550 uL phenol/chloroform was added, and fully mixed, centrifuged at 12000 rpm for 10 min.
6) Put the supernatant (450 uL) into a new 1.5 mL centrifuge tube, add equal volume of chloroform (450 uL), mix upside down, centrifuge at 12000 rpm for 10 min.
7) Drain the supernatant (300 uL) into a 1.5 mL centrifuge tube, add 0.1V NaAc (30 uL) and 0.6V isopropyl alcohol (180 uL), mix them upside down gently. Precipitation at -20 ℃ for 30 min. Centrifugation at 12000 rpm for 10 min.
8) The precipitate was washed with 500 uL 75% ethanol and centrifuged at 12000 rpm for 5 min.
9) Dissolve with 50 uL sterile water or TE.
10) DNA purity and concentration were detected by agarose gel electrophoresis.
PCR Process (one cycle)
Overview
1. Predenaturing: 98℃continues 3mins——Strands Separate
2. Denaturing: 98℃ continues 10s——Strands Separate
3. Annealing: 55℃ continues 10s——primers bind template.
4. Extnsion: 72℃ continues 25s——synthesise new strand
5. Go to step2×30
6. Extnsion: 72℃ continues 2 min
7. 4℃ HOLDING
If it is linear plasmid:
98℃ 5 min，98℃ 10 s，65℃15 s，72℃ 4 min，30 cycles，72℃ 5 min，10℃~
If it is gene:
98℃ 5 min，98℃ 10 s，68℃15 s，72℃ 2 min，30 cycles，72℃ 5min，10℃~
Then DpnI digested the annular plasmid (DpnI 1ul, Green buffer 3.5ul) at 37℃ for 5min, 4℃~
Gel Extraction
1. Cut the single target fragment from the agarose gel, place it in a clean centrifuge tube and weigh it.
2. Add three times the volume of PE solution to the gel block. If the gel weight is 0.1g, the volume is considered as 100 uL, and add 300 uL of PE solution. Using a gel cutter to cut 1% agarose gel, the weight of a single piece is about 0.06g. The actual weight of the gel is related to the gel concentration and thickness. Solizing at 15-25℃ for 5-10min at room temperature, during which the centrifugal tube is constantly gently turned up and down to ensure that the gel block is fully dissolved, if the gel block is too large, you can cut the gel block in advance.
3. Add the solution obtained in the previous step into an column CA5 (the adsorption column is placed in the collection tube), place at room temperature for 2 min, centrifuge at 12000 rpm for 30-60 s, pour away the waste liquid in the collection tube, and place the column CA5 into the collection tube.
4. Add 600 uL bleaching solution PW to column CA5 (add anhydrous ethanol before use) centrifuge at 12000 rpm for 30-60s, pour away the waste liquid in the collection tube, and put column CA5 into the collection tube.
5. Repeat the preceding operations.
6. Put the column CA5 back into the collection tube and centrifuge it at 12000 rpm for 2 min to remove the rinse solution as far as possible. The column CA5 was placed at room temperature for several minutes and dried thoroughly to prevent residual bleaching liquid from affecting the next experiment.
7. Put column CA5 into a clean centrifugal tube, add an appropriate amount of eluting buffer TB to the middle of the adsorption film, and place at room temperature for 2 min. DNA solution was collected by centrifugation at 12000 rpm for 2 min.
Plasmid construction
Plasmid construction will be carried out in the afternoon (Ice maker will be turned on at noon).
MCS2 linear Plasmid (4762 bp), required mass 0.02X4762=95.24 ng
NapA gene (2505 bp), required mass 0.04x2505 =100.2 ng
| Name | Volume |
| 5 x CE II Buffer | 4μL |
| Exnase II | 2μL |
| MCS2 linear Plasmid | 0.5μL |
| napA gene | 2μL |
| H2O | 11.5μL |
Total 20 uL then incubated with the 37 ℃, 4℃ Holding.
Plasmid transformation
1. Competent cells (e.g. DH5α Competent cell, Vazyme #C502) are thawed on ice.
2. Add 10 μL recombinant product to 100 μL competent cells, lightly flip the tube wall and mix well (do not shake the mixture), then stand on ice for 30 min.
3. After 42℃ water bath heat shock for 45 s, immediately placed on ice to cool for 2-3 min.
4. Add 900 μL SOC or LB medium (without antibiotics), shake bacteria at 37℃ for 1 h (rotation speed 200-250 rpm).
5. The LB solid medium plate with corresponding resistance was preheated in a 37℃ incubator.
6. Centrifugation was performed at 5,000 rpm for 5 min, and 900 μL supernatant was discarded. Hang the bacteria in the remaining medium and lightly coat them on a plate containing the correct resistance using a sterile coating stick.
7. Culture upside down in 37℃ incubator for 12-16 h.
Preparation of Competent cells
(1) Cell was inoculated in LB liquid medium at a volume ratio of 1:100 and cultured overnight at 28℃ until OD600 =0.5-0.6.
(2) Precool the bacteria solution on ice for 10 min. 3 mL bacterial solution was placed in a 1.5 mL sterile centrifuge tube, centrifuged at 4℃ for 5000 r/min for 5 min, and the supernatant was discarded.
(3) Add 1 mL pre-cooled 0.1 mol/L CaCl2 and gently blow and wash the suspended bacteria. Centrifugation was performed at 5000 r/min at 4℃ for 5 min. Supernatant was discarded.
(4) Add 100 μL precooled glycerol CaCl2 to resuspended precipitate and store at -70℃.
Combined transfer
(1) Prepare the membrane and sterilize it in a glass petri dish lined with filter paper. Prepare forceps, sterile water. TY medium, no resistance. Two tablets per medium.
(2) 8ml bacterial solution was centrifuged at 3500g for 5min, and thalli were collected in large quantities.
(3) Then add sterile water twice, then mix 1:1 and wash again, remove supernatant.
(4) Drop the mixed bacterial liquid on the filter membrane, each filter membrane 100 uL, culture for 48 hours, seal the petri dish with the sealing membrane.
(5) The bacteria are then washed off the membrane, into the tube.
(6) After 48 hours, dilute the cells into the plate.
