I. Plasmid extraction
Resistance (ampicillin 1:1000) was added to the previously prepared LB liquid medium, 15mL centrifuge tube was taken, and 5ml resistant liquid medium was added.
Monoclones were selected from the plate with small white Tips, and the small gun head was injected into a 15ml centrifuge tube, 30℃, 180rpm, and shaken > for 5h to see the turbidization of the liquid medium.
A clean 1.5mL EP tube was taken and 1.5ml bacterial liquid (which could be added or decreased according to the degree of turbidity of the bacterial liquid) was absorbed from the 15mL centrifuge tube. The centrifuge was 12000rpm for 3min.
Discard the supercoat and add 250ul TIANGEN plasmid mini Kits(Cat# dp103-03, Lot# 6123)P1, shake and mix thoroughly.
250ul P2 was added to gently (to avoid DNA breakage) and flipped over for 6-8 times to fully decompose the bacteria.
(The total time of this step should not exceed 5min to prevent overcracking and plasmid damage)
Add 350UL P3 liquid to the EP tube, turn it up and down gently for 6-8 times immediately, and mix it thoroughly. At this time, white flocculent precipitation can be seen. Centrifuge at 12000rpm for 3min.
(Mix immediately after P3 is added to avoid local precipitation. If there is still tiny white precipitation in the supernatant, the supernatant can be centrifuged again.)
The adsorption column CP3(before the experiment, the adsorption column was treated with equilibrium liquid BL to activate the silicon matrix membrane to the maximum extent and improve the yield;
Put the supernatant into the adsorption column CP3, and try not to absorb precipitation. A centrifuge of 12000rpm, 30-60s was centrifuged, and the waste liquid in the collection tube was poured out. Then the adsorption column was put into the collection tube.
Optional steps: Add 500ul proteinremoving liquid PD to CP3, centrifuge it at 12000rpm for 30-60s, pour away the waste liquid in the collection tube, and put the adsorption column into the collection tube.
(If the host bacteria are end A+ host bacteria TG1, BL21, HB101, JM series, ET12567, etc., this step is recommended because they contain A lot of nucleases;
If end A-host DH5, TOP10, etc., this step can be omitted)
Add 600ul bleach solution PW(anhydrous ethanol has been added) to CP3, centrifuge it at 12000rpm for 30-60s, pour away the waste liquid in the collection tube, and put the adsorption column into the collection tube.
Repeat step 9.
The centrifuge was centrifuged at 12000rpm for 2min to remove the remaining rinse solution from the adsorption column.
The CP3 column was placed into a clean 1.5mL EP tube, and 50-100ul eluent EB was added to the middle part of the adsorption film. The solution was placed at room temperature for 2min, centrifuged at 12000rpm for 2min, and the plasmid solution was collected into the EP tube.
(To increase the plasmid yield, the solution was added to the adsorption column CP3 again, placed at room temperature for 2min, centrifuged at 12000rpm for 2min, and collected into the EP tube.
The EB solution can also be preheated at 65-70℃ in advance.)
The concentration was measured and the quality of plasmid was determined.
2. Plasmid lift. Generally, plasmid lift is required to confirm the quality of plasmid before operation.
Clean conical flask was taken, 100ml LB liquid medium was added in step 1, and 10UL bacterial liquid in Step 2 was added into conical flask, shaken overnight at 30℃ and 180rpm, subject to turbidification of liquid medium.
Strain preservation: Strain preservation was performed with 15% glycerin: 350ul bacterial solution +150ul 50% glycerin was added into a 1.5mL EP tube, mixed well, and stored at -80℃.
The remaining bacterial liquid in the conical flask was centrifuged in a 50ml tube at 4℃ for 8000rpm for 5min to collect the bacterial precipitation, which could be repeated in the tube.
According to the instructions of the selected large extract kit, the extraction procedure is different for different kits.
After the extraction, the concentration was measured and the glue was run (because the molecular weight of the plasmid was generally large, agarose glue with low concentration and big marker were used).