Experimental principle
After electrophoresis, the DNA fragments cleaved by restriction enzymes are separated according to molecular weight and arranged in agarose gel. The gel where a specific DNA band is located can be cut off, heated or melted with special reagents. The gel is used to dissolve the DNA back into the solution, and then the ethanol-digested fragments can be obtained by ethanol precipitation or passing through the column.
Experimental reagent
1. Electrophoresis buffer
2. Fluorescent dyes
3. Electrophoresis grade agarose powder
4. 10´ loading buffer
5. DNA molecular weight standard (DL2000)
6. DNA gel recovery kit
experiment apparatus
Vortex mixer
2. Micro pipette sampler
3. Pipette tips
4. 1.5ml microcentrifuge tube
5. Double-sided microcentrifuge tube rack
6. Desktop centrifuge
7. Agarose gel electrophoresis system
8. Microwave oven
9. Constant temperature water bath
Experimental procedure
1. Prepare TAE electrophoresis buffer (10´storage solution), 10´sampling buffer, 1.5ml centrifuge tube into aluminum lunch box (sterilization), pipette tip into corresponding tip box (sterilization) ).
2. Operation steps
1) Prepare 1% agarose gel with 1´TAE. DNA is digested with a suitable enzyme.
2) Select two sample wells on the gel, add a small amount (10μl) and a large amount (50μl) of DNA digestion products, and electrophorese.
3) After electrophoresis, use a blade to cut the lane containing a small amount of DNA digestion product (generally choose to locate at the edge of the gel), find the target DNA band under the UV lamp, and use the blade to cut a small opening at the lower and upper edges of the band mark. Note: The gel containing a large amount of DNA digestion products does not need to be added with fluorescent dyes or ultraviolet light!
4) Align the marked gel strip with the unstained gel in situ, estimate the position of the DNA digestion product on the unstained large gel according to the marking on the small gel strip, and cut the DNA product in the large gel with a blade Transfer the gel (as much as possible) to a clean 1.5ml microcentrifuge tube. After weighing, calculate the weight of the glue.
5) Purify and recover the target fragment according to the instructions of the DNA rapid purification / recovery kit.
Purification / recovery kit composition:
Solution A: 6M NaClO4, 0.03M NaAc, pH5.2, a small amount of phenol red;
Solution B: 3M NaAc;
Solution C: add 35ml absolute ethanol when using;
Solution D: TE buffer.
The glue recovery steps are as follows:
a. Put the cut tape into a 1.5ml centrifuge tube, and add solution A according to the ratio of 1: 3 (weight of tape: volume of solution A);
b. Dissolve in water at 50 ℃ for 10min, the tape needs to be completely dissolved, and can be shaken to help dissolve 2 to 3 times. After dissolution, add 15μl of solution B at room temperature and mix thoroughly;
c. Place the solution in a spin column, let stand 2min, centrifuge at 10000rpm for 20s;
d. Pour off the liquid, add 500μl of solution C to the spin column and centrifuge at 10000rpm for 20s, discard the liquid and repeat this step once;
e. Centrifuge at 12000rpm for 1min, spin off the remaining liquid to remove residual ethanol;
f. Place the centrifuge column in a new centrifuge tube, open the centrifuge tube cover at room temperature for 5 to 10 minutes to allow the ethanol to evaporate;
g. Add 20 ～ 30μl of solution D (50 ℃ water bath before use) and let stand for 2min;
h. Centrifuge at 12000rpm for 1min. The solution at the bottom of the tube is the required DNA. Store the DNA at -20 ℃ for long-term storage.
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