This kit supports
50-2,000 ng of gDNA or cDNA. For input amount exceeds 2,000 ng, split the
sample into multiple reactions to maintain amplification efficiency.
The main peak of
PCR products by using this kit is ~270 bp. PE150 sequencing is recommended for
high-quality coverage.
A minimum of 0.3
Gb is recommended to detect clones at 0.01% abundance with an input of 200 ng.
Increase data volume to enhance sensitivity for low-frequency clones.
Yes. This kit
includes IG Primer Mix and TR Primer Mix with gene-specific primers provided in
separate tubes, allowing flexible combination in a single amplification
reaction. With its high sensitivity, the kit meets the requirements for MRD
monitoring technology development and clinical applications, making it ideal
for low-frequency variant detection scenarios.
√ If the sample is a co-extracted total nucleic acid, quantification of RNA and DNA should be performed separately using Qubit, and library amplification should be carried out according to the recommended cycles in the manual.
√ During the experiment, RNase-free consumables must be used, and the operating environment should be free of RNase contamination, as this can affect the efficiency of RNA reverse transcription.
√ If the nucleic acid sample contains excessive amounts of chelating agents, guanidine salts, phenol, proteins, ethanol, or other impurities, it can impact the activity of RNA reverse transcriptase and the efficiency of DNA enzymatic digestion. It is recommended refer to purify the nucleic acid samples using 2X NadPrep SP Beads, replace the elution reagent with Nuclease Free Water, and then proceed with library preparation.
√ For samples of lower quality, consider increasing the number of PCR amplification cycles appropriately.
If the nucleic acid samples contain high concentrations of EDTA or have an excessively high pH, it can affect the efficiency of DNA digestion. It is recommended to purify nucleic acid samples using 2X NadPrep SP Beads, replacing the elution reagent with Nuclease Free Water before proceeding with library preparation.
This module can tolerate nucleic acid samples containing 6 μL of EDTA (1 mM), resulting in a final concentration of 0.3 mM in the cDNA first-strand synthesis system. At this concentration, RNA reverse transcription inhibition is minimal, and the efficiency of enzymatic digestion is unaffected. Before library preparation, confirm the sample solvent. If it exceeds the recommended EDTA threshold, it is necessary to purify nucleic acid samples using 2X NadPrep SP Beads, replacing the elution reagent with Nuclease Free Water before proceeding with library preparation.
When co-prepare RNA & DNA libraries from mixed nucleic acid samples, a higher proportion of DNA samples results in larger fragments with enzymatic digestion. Adjustments can be made according to the expected insert fragment lengths and recommended time of enzymatic fragmentation in the manual.
Libraries prepared with this module can be used in conjunction with NEX-t Panel v1.0 and NadPrep ES Hybrid Capture Reagents. This significantly shortens the time required for pathogen-targeted sequencing and simplifies experimental procedures.
Figure 1. Library yield and capture performance of DNA samples using different library preparation protocols. A. Library Yield; B. Mappability.
Note: The sample originated from 50 ng of Human Genomic DNA Standard (Promega, G1521). Sequencing mode was Illumina NovaSeq 6000 platform, PE150.