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Tips for Improving Glue Recovery

1. Increase the sample load during electrophoresis.

2. Use freshly prepared electrophoresis buffer.

3. When cutting glue, try to cut only the glue with strips to reduce the volume of glue cutting: do not need glue with few purpose fragments, otherwise it will affect the recovery rate.

4. After melting two or more pieces of glue, use a tube no matter how large the volume is and transfer it to the same column.

5. The solution added in the sol can be a little more, which is more conducive to the binding of DNA to the membrane, but generally do not exceed 750ul.

6. The key to gel recovery is to bind DNA to the column through the salt concentration, acidity (charge) and hydrophobicity of the solution in the column. Therefore, if the pH of the electrophoresis buffer is too high, 10ul (pH 5.0, 3mol/L NaAC) can be added to the sol; in order to better intercept DNA molecules on the membrane, 30% isopropanol can be added to heat into the liquid after dissolving the glue.

7. Before adding the eluent, leave the column at room temperature for a few minutes (about 10 minutes) to fully evaporate the ethanol.

8. Finally, add less eluent to minimize the recovery volume. Generally, 30-50μl eluent is used for elution (not too little, otherwise it will not be able to wet the membrane, which is not conducive to elution); the elution droplets are in the center of the membrane, to fully elute the DNA bound to the membrane.

9. After adding the eluent, it can be eluted in a water bath of 55 degrees for 5 minutes or placed in a water bath of 50 degrees for more than 10 minutes, or sealed with a parafilm at 4 degrees overnight, and then centrifuged for recovery the next day, the effect is good.

10.Add the centrifuged eluate back to the adsorption column and centrifuge again.

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Detailed methods and procedures for PCR product recovery

1. Ordinary rubber recycling

If you want to recover the glue, it is best to use a kit, which is convenient and has a slightly higher recovery rate. If you really need to recover it manually, you can add 3 times the volume of TE after cutting the glue. After melting in a water bath, phenol, phenol/chloroform are extracted cleanly, and ethanol is precipitated. That’s it.

2. DNA recovery from low melting point gels

Purification of DNA Fragments Add TE (10 mmol/l Tris-HCl pH8.0, 0.1 mmol/l EDTA) equal to the volume of the gel, and place in a 65°C water bath for 5 minutes to dissolve the gel completely.

After it was brought to room temperature, an equal amount of phenol (saturated with TE, TE was sealed in the upper layer, and the lower layer of phenol was removed) was added, and the mixture was gently mixed (no mixing required), and centrifuged at 12,000 rpm for 3 minutes. Repeat 1-2 times.

Take the supernatant, add 0.1 volume of 3mol/L sodium acetate (pH 5.2) and 2.5 times volume of absolute ethanol to carry out ethanol precipitation. Dissolve the purified DNA with an appropriate amount of TE, measure the content, and prepare for use (it can be used for target gene structure analysis, probe preparation, etc.).

3. PCR recovery with good amplification specificity

If the specificity of PCR amplification is good, it is just a simple purification and recovery of the PCR product. You can add 50ug/ml proteinase K to the PCR product, 37 degrees for 1h, extract once with phenol/chloroform, extract once with chloroform, and add 0.1 volume of the supernatant. The sodium acetate was recovered by precipitation with 2.5 volumes of absolute ethanol.

Related products:

https://www.foreivd.com/pcr-purification-kit-2-product/

https://www.foreivd.com/blood-superdirecttm-pcr-kit-edta-product/


Post time: Sep-24-2022