Common problems during extraction
Nucleic acid extraction methods include column method and magnetic bead method, and the extraction steps are basically similar into cracking, washing and elution. Nucleic acid extraction requires attention to a lot of details, a mistake will lead to the failure of the experiment. The problems are mainly low product yield, degradation or reagent residue
1. Nucleic acid yield is low or not extracted
Solution:
1> Select the extraction reagent that matches the sample or use the correct extraction procedure.
2> The extraction reagent should be stored according to the storage method in the instructions, avoid the decomposition of the reagent and affect the extraction effect.
3> The ratio of sample to reagent usage should be controlled in an appropriate range to avoid exceeding the cracking capacity
4> Crushing and grinding (homogenate, cracking) must be sufficient, and insufficient grinding (homogenate, cracking) is easy to cause less release of nucleic acid, resulting in low extraction efficiency. If this happens, the grinding (homogenizing, cracking) time can be extended appropriately.
5> Ethanol must be added to the washing buffer before use, and low ethanol content will affect the washing effect and lead to low nucleic acid yield.
6> The amount of eluent used too little in the elution step, would resulting in inadequate elution and low concentration. Too much eluent will dilute the concentration of nucleic acid, resulting in a lower concentration. So it’s important to choose the right amount of eluent.
7> When the magnetic bead method is used to extract nucleic acid, the magnetic bead must be fully mixed during elution so that the magnetic bead is in a completely dispersed state, otherwise the elution effect will be affected and the extraction efficiency will be reduced.
2. Degradation in extraction experiment
1> We recommend fresh samples or samples that have been thawed only once; avoid repeated freeze-thaw samples,
2> For long-term preservation of samples, appropriate preservation conditions should be selected to avoid nucleic acid degradation caused by improper preservation.
3> The operation time is too long, or the ambient temperature is too high, resulting in nucleic acid degradation;
4> When the temperature is too high during elution, the nucleic acid may degrade
5> We’d better have fresh gel and electrophoresis solution, The old electrophoretic solution causes electroosmosis, excessive current, heat and so on, which leads to the degradation of nucleic acids, especially RNA
6> The electrophoresis tank should also be cleaned with RNase-free water before electrophoresis to avoid RNase residue resulting in RNA degradation.
3. Low purity
1> The proportion of sample size and reagent used is not right, the grinding (homogenization, cracking) is not enough, resulting in excessive adsorption of protein coating or magnetic beads;
2> When the supernatant was absorbed, the precipitate was accidentally absorbed after centrifugation.
3> The adsorption column or magnetic beads are contaminated with reagents during operation, especially during elution
4> Inadequate washing results in the residue of ion or protein.
5> Insufficient drying time after washing leads to residual ethanol and inhibits downstream experiments
1. Nucleic acid yield is low or not extracted
Solution:
1> Select the extraction reagent that matches the sample or use the correct extraction procedure.
2> The extraction reagent should be stored according to the storage method in the instructions, avoid the decomposition of the reagent and affect the extraction effect.
3> The ratio of sample to reagent usage should be controlled in an appropriate range to avoid exceeding the cracking capacity
4> Crushing and grinding (homogenate, cracking) must be sufficient, and insufficient grinding (homogenate, cracking) is easy to cause less release of nucleic acid, resulting in low extraction efficiency. If this happens, the grinding (homogenizing, cracking) time can be extended appropriately.
5> Ethanol must be added to the washing buffer before use, and low ethanol content will affect the washing effect and lead to low nucleic acid yield.
6> The amount of eluent used too little in the elution step, would resulting in inadequate elution and low concentration. Too much eluent will dilute the concentration of nucleic acid, resulting in a lower concentration. So it’s important to choose the right amount of eluent.
7> When the magnetic bead method is used to extract nucleic acid, the magnetic bead must be fully mixed during elution so that the magnetic bead is in a completely dispersed state, otherwise the elution effect will be affected and the extraction efficiency will be reduced.
2. Degradation in extraction experiment
1> We recommend fresh samples or samples that have been thawed only once; avoid repeated freeze-thaw samples,
2> For long-term preservation of samples, appropriate preservation conditions should be selected to avoid nucleic acid degradation caused by improper preservation.
3> The operation time is too long, or the ambient temperature is too high, resulting in nucleic acid degradation;
4> When the temperature is too high during elution, the nucleic acid may degrade
5> We’d better have fresh gel and electrophoresis solution, The old electrophoretic solution causes electroosmosis, excessive current, heat and so on, which leads to the degradation of nucleic acids, especially RNA
6> The electrophoresis tank should also be cleaned with RNase-free water before electrophoresis to avoid RNase residue resulting in RNA degradation.
3. Low purity
1> The proportion of sample size and reagent used is not right, the grinding (homogenization, cracking) is not enough, resulting in excessive adsorption of protein coating or magnetic beads;
2> When the supernatant was absorbed, the precipitate was accidentally absorbed after centrifugation.
3> The adsorption column or magnetic beads are contaminated with reagents during operation, especially during elution
4> Inadequate washing results in the residue of ion or protein.
5> Insufficient drying time after washing leads to residual ethanol and inhibits downstream experiments
