How to Use Micro-Volume Spectrophotometer to Guide the Improvement of Nucleic Acid Extraction Results

Introduction

Nucleic acid extraction is a crucial step in molecular biology experiments, and its quality directly affects the success or failure of downstream experiments (such as PCR and sequencing). The micro-volume spectrophotometer, with its characteristics of high sensitivity, rapid detection, and support for low volume samples, can evaluate the purity and concentration of nucleic acids in real time, providing accurate data support for optimizing the extraction process. In this article, we will introduce how to improve the quality of nucleic acid extraction based on the detection results of micro-volume spectrophotometer.

Materials and Methods

We used a 32-channel nucleic acid extractor and a genomic nucleic acid extraction kit to extract nucleic acids from whole blood samples. The extraction process is as follows: We used the genomic nucleic acid extraction kit. According to the instructions, relevant reagents for lysis, washing, elution, etc. were sequentially added into a 96-well deep plate. Subsequently, 200 μL of the whole blood sample and 15 μL of proteinase K were placed in well position 1 of the 96-well plate, and the magnetic beads were placed in well position 2 of the 96-well plate. According to the usage method shown in the instructions, the operation steps of Program 1 were compiled and run.

Figure 1: Program 1

After the program is completed, take out the deep well plate, and the nucleic acid extractor runs the ultraviolet disinfection program.

Use Four E's Scientificmicro-volume spectrophotometer to detect the samples in the elution wells, and the detection results are summarized as shown in Figure 2.

Figure 2

Under normal circumstances, approximately 5-8 μg of nucleic acid can be obtained from a 200 μL whole blood sample. For pure nucleic acid, the A260/A280 ratio should be higher than 1.8, and the A260/A230 ratio should be higher than 2.0. The extraction results shown in Figure 2 indicate that the nucleic acid concentration in the eluent is low and the purity is also low, which fails to meet the requirements of the next experimental step. By using micro-volume spectrophotometer to detect the residual amount of nucleic acid on the magnetic beads after another elution, it is found that the reason for the low extraction concentration is that both the lysis reaction and the elution process are insufficient. Therefore, the extraction program is changed, and the changed program is shown in Figure 3. Run the program in Figure 3:

Figure 3: Program 2

After the program is finished, take out the deep well plate, and the nucleic acid extractor runs the ultraviolet disinfection program. Use the micro-volume spectrophotometer to detect the samples in the elution wells. The detection results are summarized as shown in Figure 4.

Figure 4

The nucleic acid concentration in the elution increased significantly, and the purity of the extracted product, as indicated by the A260/280 and A260/230 ratios, improved notably, making it basically suitable for PCR experiments. However, there was still contamination from the lysis buffer or washing solution. The extraction program was modified again, and the revised program is shown in Figure 5. The procedure in Figure 5 was then executed:

Figure 5: Program 3

After the program is completed, remove the deep-well plate, and the nucleic acid extractor will initiate the UV disinfection program. Use the micro-volume spectrophotometer to measure the samples in the elution wells. The detection results are summarized in Figure 6.

Figure 6

The extracted product has a high concentration and good purity. It can be stored frozen at -20°C, or directly used for downstream experiments.

Conclusion

Without the kit being adapted to the machine, there are often problems when directly using the procedures within the kit on the automated extraction equipment. The micro-volume spectrophotometer can be used to detect the results of nucleic acid extraction in real time, analyze the reasons for extraction failure and make improvements, and then guide and adjust the extraction procedure, ultimately achieving excellent nucleic acid extraction results.