1. Prepare an agarose EB gel and electrophoresis to separate DNA fragments. Any type or grade of agarose can be used.

We strongly recommend using fresh TAE/TBE electrophoresis buffer. Do not reuse electrophoresis buffer, as old electrophoresis buffer will increase the pH and decrease the yield of DNA recovery;

2. After electrophoresis for a sufficient period of time, carefully cut off fragments of the desired DNA under a UV lamp. And remove as much excess gel as possible.

Note: Do not expose the DNA under the UV lamp for more than 30 seconds, and also be sure to wear protective glasses when operating under the UV lamp.

3. Weigh the empty centrifuge tube, cut off the gel with the target fragment in a 1.5 ml centrifuge tube and weigh it, find out the weight of the gel block and approximate its volume. In general, the density of the gel is 1g/ml, so the relationship between the volume of the gel and its weight can be converted as follows: the weight of the gel sheet is 0.2g, then its volume is 0.2ml; add equal times the volume of the gel Binding Buffer, the mixture is placed in the 55 ℃ ~ 65 ℃ water bath for 7min to the complete melting of the gel, which is mixed every 2-3 minutes;

Important note: the gel was cut off with the target fragment in a 1.5ml centrifuge tube and weighed to determine its volume. p>

Important note: After the gel is completely dissolved, pay attention to the pH value of the gel-Binding Buffer mixture. The yield of DNA will be greatly reduced if its pH is greater than 8. Observe the color of the mixture, if it is orange or red, add 5 μl of sodium acetate at a concentration of 5 M and a pH of 5.2 to lower its pH. After this adjustment, the color of the mixture will return to its normal light yellow color. In general, the pH of the gel-binding buffer mixture will not be elevated when fresh electrophoresis buffer is used;

4. Transfer 700 μl of the DNA-agarose solution to a HiBindTM DNA column and mount the column in a clean 2 ml collection tube and centrifuge for 1 min at room temperature at 10,000 ×x g centrifugation for 1 min and discard the liquid.

A HiBind DNA column can hold up to 700 μl of solution. If the volume of the DNA-agarose mixture is greater than 700 μl, you can transfer 700 μl of solution to the column first, and after centrifugation, continue to add the remaining solution to the column. However, each HiBindTM column can bind a maximum of 25-30 μg of DNA. if a larger yield is expected, add the samples to the appropriate number of columns separately.

5. Put the column back into the collection tube and add 300μl of Binding Buffer to the HiBind DNA column; centrifuge the column at 10,000×g for 1 minute at room temperature and discard the filtrate; this is a critical step, so don't overlook it.

6. Put the column back into the collection tube, add 700 μl of SPW Wash buffer to the HiBind DNA column, and centrifuge at 10,000 × g for 1 minute at room temperature to discard the filtrate; Note: SPW Wash buffer must be diluted with anhydrous ethanol according to the bottle labeling requirements before use.

7. Replace the column back into the collection tube and add 700 μl of SPW Wash buffer to the HiBind DNA column, and centrifuge at 10,000 × g for 1 min at room temperature to remove the filtrate;

8. Dispose of the liquid, re-sleeve the empty column back to the collection tube, and centrifuge at 10,000 × g for 1 min to shake off the liquid residue from the column matrix. of the liquid.

This step removes residual ethanol from the column matrix, and should not be skipped - it is important to get good DNA yields.

9. Mount the column on a clean 1.5 ml centrifuge tube, add 30-50 μl of eluent or sterilized water on the column membrane, centrifuge for 1 minute at 10,000 x g. The solution in the centrifuge tube is the purified DNA product, store at -20 degrees.

Residual DNA can be eluted out if you elute again, but then the concentration will be lower.

DNA yield and quality:

Dilute the purified sample by a certain number of times, and then measure the light absorption value at 260nm and 280nm respectively, and the concentration of the recovered DNA can be calculated according to the following formula: DNA Concentration=Light Absorbance 260×50×dilution times μg/ml

Fragment with a length of more than 500bp can be purified with a yield of 80%. can be purified to get 80% yield. while bands of 50bp~500bp can achieve 55%~80% recovery. The ratio of (light absorption 260/light absorption 280) is a marker of nucleic acid purity. If this value is 1.8, it means that the purity of the nucleic acid is >90%. On the other hand, if the yield of the purified product is low, the concentration of the product can be estimated by agarose EB electrophoresis.