Nucleic Acid Extraction Troubleshooting

 
1. Low yield or no yield
Possible solutions:
· Ensure that the reagent or kit selected for extraction is a good fit for your sample(s) and desired downstream applications.
· Always store the kit and all reagents according to the instructions to avoid degradation of the reagents and reduced extraction effectiveness.
· For optimal cell lysis and nucleic acid extraction, ensure that you are using the appropriate amount of reagents for your sample, and do not exceed the lysing capacity.
· For best extraction results, ensure that your sample(s) have been homogenized and/or lysed sufficiently. Insufficient lysis or homogenization is a common cause of low yield. If necessary, increase homogenization or lysis of cells for better yields.
· Always add ethanol to the wash buffer before use. Low ethanol content can affect the washing effectiveness and lead to low nucleic acid yields.
· Ensure that you are using an appropriate amount of eluent in the elution step. Using too little eluent can lead to low yields with low concentrations. Using too much eluent can dilute the concentration of product, making it difficult to use in certain downstream applications.
· When using magnetic bead extraction kits, ensure that the magnetic beads are fully mixed during the elution step for complete retrieval of the extracted products. If beads are not mixed well, this can negatively affect the extraction yield.
 
2. Product Degradation
Possible Solutions:
· Whenever possible, always use fresh samples or samples that have only been frozen and thawed once. Avoid repeated freezing and thawing of samples to preserve sample integrity.
· For long term storage and preservation of samples, use appropriate conditions to avoid as much nucleic acid degradation as possible. Avoid repeated freezing and thawing of samples to preserve sample integrity.
· If the experimental time is too long, or the ambient temperature is too high, this can result in nucleic acid degradation.
· If the temperature is too high during elution, this can also result in nucleic acid degradation
· Whenever possible, use freshly prepared gels and electrophoresis solutions. Old electrophoresis solutions can cause electroosmosis, excessive current, excessive heat, etc, which can lead to degradation of nucleic acids, especially RNA products.
· When working with RNA, the electrophoresis tank should be cleaned with RNase-free water before electrophoresis to avoid any RN degradation from RNase residue.
 
3. Low purity
Possible Solutions:
· The amount of sample used with the kit or reagent is incorrect, or there is insufficient homogenization or lysis, which results in excessive adsorption of the protein coating or magnetic beads.
· Some precipitate was accidentally pipetted with the supernatant after centrifugation.
· The spin column filter or magnetic beads were contaminated during the experiment. This is especially common during the elution step.
· Inadequate washing can result in ion or protein residue in the products.
· Insufficient drying time after washing steps can lead to residual ethanol still being present, which often inhibits downstream experiments.