Team:XHD-Wuhan-B-China/Protocol
PCR
1) Prepare Max system: take Max solution 15ul, f (front-end primer DNA fragment) 1ul, R (back-end primer) 1ul, dna1ul (dilute 5ng / UL), deionized water 12ul
2) Preheat at 98 ℃ for five minutes to split the double chain
3) Start cycle
Preheat at 98 ℃ for 10s and split the double chain
Primer binding at 55 ℃ for 15s
Replication and extension at 72 ℃ for two minutes and 20 seconds
4) Repeat the cycle for 30 times
5) 72 ℃ for five minutes to ensure the extension time and obtain complete coupling
6) Store at 4 ℃
7) DpnI digestion template circular plasmid
Add DpnI 1ul and green buffer 3.5ul and react at 37 ℃ for five minutes. This step is used to prevent false positive transformation results.
8) Recovery of linear products by gel running
DNA electrophoresis
1) Put the cleaned and dried glue making plate into the glue making tank and place it horizontally on the workbench
2) Prepare 1.0% agarose gel: weigh 0.3g agarose in 30ml 1 ×TAE, completely dissolve agarose particles in a microwave oven, add DNA dye when cooling to warm heat, mix well and pour glue
3) carefully remove the comb after the gel has set.
4) mix the DNA sample with 3.5ul loading buffer(if did’t use green buffer), and then add the sample into the sampling hole in turn
5) put the gel preparation plate into the electrophoresis tank, add electrophoresis buffer, turn on the electrophoresis instrument, adjust the voltage to 80V, and make the nucleic acid sample swim to the positive electrode (note that the sampling hole is at the negative end of the electrophoresis tank)
6) after electrophoresis, cut off the power supply, take out the gel, observe the electrophoresis results on the UV transmission instrument, take photos and record, and select the strip to cut the glue.
Recover DNA fragments from gel
1) The agarose gel containing the target DNA fragment was cut off under UV light and transferred to a 1.5 ml centrifuge tube.
2) Weigh the weight of the gel and add 3 times the volume of buffer g (converted into 1ul gel volume per 1mg gel). For example, 300 UL buffer g should be added to 100 mg gel.
*If the concentration of gel is greater than 2%, 6 times the volume of buffer g shall be added.
Instrument used: electronic balance - > more accurate
Add buffer g to dissolve the gel
3) Put the centrifuge tube containing gel in a water bath at 50 ℃ until the gel is completely dissolved (about 5-10 minutes).
*During the process of sol, turn over the centrifuge tube several times every 2-3 minutes to help the gel dissolve and observe whether the gel dissolves completely.
*The dye added in buffer g can help to observe whether the gel is completely dissolved. At the same time, it can indicate the pH value. If the solution turns purple red, 10 UL 3 M sodium acetate (pH 5.0) should be added to restore the solution to orange yellow, otherwise it will affect the binding of DNA to the purification column.
4) Transfer the sol solution to the nucleic acid purification column (the nucleic acid purification column is placed in a 2ml centrifuge tube). Centrifuge at 12000 rpm for 30 seconds, discard the filtrate in 2ml centrifuge tube, and place the nucleic acid purification column back into 2ml centrifuge tube.
*If the volume of sol solution is greater than 800 UL (e.g. DNA recovery from 200 mg gel), it shall be centrifuged through the column twice
*The filtrate does not need to be completely discarded. To avoid the pollution of the filtrate adhered to the nozzle of the centrifugal pipe to the centrifuge, the 2ml centrifugal pipe can be buckled and patted on the paper towel once.
5) Add 500 UL buffer ws to the nucleic acid purification column, cover the tube cover, and centrifuge at 12000rpm for 30 seconds. Discard the filtrate in the 2ml centrifuge tube and place the nucleic acid purification column back into the 2ml centrifuge tube.
*This step is to remove residual trace agarose molecules. If the recovered DNA is not used for sequencing, external transcription or microinjection experiments, this step can be omitted.
6) Add 700 UL buffer WG, cover the tube, and centrifuge at 12000 rpm for 30 seconds.
*If the recovered DNA is used for salt sensitive experiments, such as flat end ligation experiment or direct sequencing, it is recommended to centrifuge after adding buffer WG and standing at room temperature for 2-5 minutes.
*Confirm that absolute ethanol has been added to buffer WG.
7) Repeat step 6) once.
8) Discard the filtrate in 2ml centrifuge tube, put the nucleic acid purification column back into 2ml centrifuge tube, and centrifuge at 14000 rpm for 1 minute.
*If the centrifugation speed is less than 14000 rpm, centrifuge at the highest speed for 2 minutes.
*Do not omit this step, otherwise the purified nucleic acid may be mixed with ethanol and affect the subsequent experimental effect.
9) Discard 2 ml centrifuge tube, place the nucleic acid purification column in a clean 1.5 ml centrifuge tube (provided by the kit), add 25 ~ 30 UL buffer TE or deionized water in the center of the membrane of the purification column, cover the tube cover, place it in the chamber for 1 minute, and centrifuge at 12000rpm for 30 seconds to elute DNA.
*If DNA is washed with several ionic water, ensure that the pH of the deionized water used is 7.0 ~ 8.5, otherwise it will affect the elution efficiency of DNA.
10) After discarding the purification column, the eluted DNA can be used in various molecular biology experiments immediately; Or store DNA at - 20 ℃.
Transformation (calcium conversion)
The whole process needs to be carried out in ultra clean platform and low temperature environment!
1) Sterilize the ultra-clean table for 20min. Take T1 competent cells of Escherichia coli (- 80 ℃) and put them on ice
2) Transfer 25ul of competent cell suspension into a 1.5ml centrifuge tube, add 8ul of connecting product and put back on ice, bath for 30min.
3) Heat shock in 42 ℃ water bath for 90s, quickly transfer the tube to ice bath for 2min, and do not shake away in the process
The plasmid was transformed into cells by heat shock
4) Add 500ul LB liquid medium into each centrifuge tube, gently suck it with a gun, mix it, and
culture in a shaker at 37 ℃, 200 rpm for 30min to resuscitate the bacteria.
5) Suck 100ul bacterial solution, add it to LB solid medium plate containing corresponding antibiotics, and use coating rod to apply evenly and incubate overnight at 37 ℃
The solid medium is liquid medium, add 1.2% ~ 1.5% agarose.