The kit is optimized for the co-extraction of RNA and DNA, and enables the simultaneous extraction of RNA and DNA from pathogens, including viruses, bacteria, and fungi, from the same sample. The final products are nucleic acids mixture. If only RNA or DNA is required for downstream applications, an appropriate nuclease treatment can be performed after extraction according to the specific experimental requirements. The treatment conditions should be established based on the downstream application and the nuclease manufacturer's instructions and validated experimentally. 

• Samples: Improper sample storage, repeated freeze-thaw cycles, or prolonged storage may compromise nucleic acid stability and reduce yield. It's recommended to store and handle samples according the conditions specified in this user manual, avoid repeated freeze-thaw cycles whenever possible, and prioritize the use of fresh samples. Special sample types should be pretreated as specified. For example, viscous samples (e.g., sputum) should be pre-liquefied according to their viscosity, while whole blood samples should be pretreated by adding an equal volume of 1X PBS or sterile saline. For the other sample types not listed in this user manual, please contact technical support (support@njnad.com) or use a validated pretreatment method. In addition, a low pathogen load in the sample may also result in low or undetectable nucleic acid yield. For these samples, extraction may be performed more than once, and a smaller elution volume may be used to increase the nucleic acid concentration.
• Reagents: Precipitates in Patho-Lysis Buffer and Patho-Binding Buffer that have not fully dissolved may affect nucleic acid extraction performance. Inspect these reagents for precipitates prior to use. If present, heat the buffers in a water bath or dry bath at 56°C until the precipitates have completely dissolved. Vortex to mix thoroughly and allow them to equilibrate to room temperature before use.
• Operations: 1) Insufficient sample lysis may result in incomplete nucleic acid release. Follow the lysis procedure specified in this user manual, ensuring thorough mixing and compliance with the specified lysis time and operating conditions. 2) Improper handling during washing may result in beads loss. When washing with Patho-Wash Buffer 1 (with absolute ethanol added) and Patho-Wash Buffer 2 (with absolute ethanol added), avoid aspirating or disturbing the beads. Dry the beads for approximately 5 min and avoid over-drying, as this may reduce subsequent nucleic acid recovery.
• Temperature: If the actual temperature during lysis and incubation does not reach the set temperature, lysis efficiency may be affected. When using a water bath or dry bath, make sure the actual temperature has reached 56°C as specified in this user manual before incubation, and maintain the specified incubation time.

The kit combines mechanical and chemical lysis for sample processing and can be used to extract nucleic acids from micro-organisms with thick cell walls, including fungi (e.g., yeasts and Candida spp.) and Gram-positive bacteria. For these pathogen types, follow the specified lysis procedure to ensure efficient nucleic acid release. Actual extraction performance may vary with sample type, pathogen load, and sample handling etc. 

1) Insufficient nucleic acid yield: Low nucleic acid concentration may not meet the requirements of downstream applications. Check sample quality, pretreatment, lysis, beads binding, and elution. If necessary, interpret the results in conjunction with the internal, positive, or negative control results.

2) Poor nucleic acid purity: Residual wash buffer or other impurities in the elute may interfere with downstream applications. Ensure that wash buffers are prepared correctly, complete all required washing steps, and thoroughly remove residual liquid before elution.

3)  Nucleic acid degradation: Improper sample storage, repeated freeze-thaw cycles, or nuclease contamination during the experiment may cause nucleic acid degradation. Store samples and purified nucleic acids as specified in this user manual and avoid repeated freeze-thaw cycles. Use nuclease-free consumables and reagents during the entire workflow, and take precautions to prevent DNase and RNase contamination.

4) Abnormalities or contamination during the workflow: Use appropriate internal, positive, and negative controls according to the specific downstream application to help determine whether the nucleic acid extraction and subsequent experimental procedures have proceeded as expected and to investigate potential contamination.

Yes. However, sample quality may affect DNA extraction performance. In general, fresh blood samples stored under appropriate conditions are more likely to get high-quality genomic DNA. Samples that are of poor quality or have been stored for prolonged periods may result in a low DNA yield and purity. For such samples, the extraction performance can be optimized by increasing the amount of Proteinase K or extending the lysis time. 

Yes. For a 100 μL concentrated blood sample, refer to the extraction procedure for a 200 μL blood sample in this user manual. If the actual sample volume differs, adjust the sample pretreatment procedure and the volumes of the relevant reagents according to the instructions for different sample volumes provided in this manual. 

The recommended standard input volume for this kit is 200 μL. For blood samples of other volumes, refer to the Protocol section of this user manual for specific processing requirements for different sample volumes. For blood input volumes of 200-1,000 μL, if a Red Blood Cell Lysis Buffer is unavailable, the validated pretreatment procedure can be used. For example, add 3X volumes of 1X PBS to the blood sample (e.g., add 900 μL of 1X PBS to 300 μL of blood sample). Mix thoroughly by inversion and centrifuge at 2,000 × g for 5 min. Carefully remove and discard the supernatant using a pipette. Add 200 μL of 1X PBS and vortex to mix thoroughly, then proceed with the Genomic DNA Extraction Workflow. 

• Low input amount: If the total nucleic acid input is extremely low (< 1 ng), a reduced library yield is expected and considered normal; subsequent downstream steps can proceed.

• RNase contamination: Use RNase-free consumables throughout the workflow and perform all the operations in RNase-free conditions. RNase contamination will reduce reverse transcription efficiency and low library yield.

• Low sample purity: Residual contaminants in RNA (e.g. chelators, guanidine salts, phenol, ethanol) may inhibit the activity of reverse transcriptase and DNA enzymatic digestion. It is recommended to purify nucleic acid samples according to Step 7: Post-amplification Cleanup using 2X NadPrep SP Beads and elute in Nuclease Free Water.

• Poor sample quality: For low-quality nucleic acid samples, follow the recommendations to increase the no. of PCR cycles appropriately.