NadPrep EZ DNA Library Preparation Kit v2

The kit can be used after being placed on the table at room temperature overnight without UV light irradiation. NadPrep EZ DNA Library Preparation Kit v2 hasa stable performance even at room temperature 12 h.

NadPrep EZ DNA Library Preparation Kit v2 is recommended to be frozen and thawed within 15 times. In order to ensure the data quality, it is recommended to use it in sub packaging. Please mix well before sub packaging.

In order to ensure the data quality, it is best to prepare the library within the scope recommended in the user manual. If there are special experimental needs, it can also be adjusted appropriately. The libraries prepared fromdifferent input DNA areshown as follows:

Experimental conditions: Human Genomic DNA Male (Promega, catalog # G1471) was fragmented with different input DNA, with 25 min of the enzymatic digestion time. NadPrep Universal Stubby Adapter (UDI) Module was used together for library preparation with single-sided selection mode. The library yield and size meet the requirements of sequencing.

• Loss caused by double-sided selection after fragmentation

Experimental conditions: 500 ng of Human Genomic DNA Male (Promega, catalog # G1471) was fragmented by using different enzymatic digestion times, and the procedures were conduct at 25℃ for 15, 20, 25 and 30min, respectively, then65℃ for 30 min. At the end of the fragmentation procedures, according to Appendix II 1.1 of the user manual, double-sided selection was used. After selection, Nuclease Free water was used to elute for subsequent adapter ligation.

This step of selection can make the fragmented samples more concentrated, thus leading to high efficiency of subsequent adapter ligation, but the sample loss is relatively high (about 60-70%), which is suitable for the library preparation of sufficient DNA. It is recommended that input DNA is ≥200 ng

• Loss caused by double-sided selection after ligation

Experimental conditions: 100 ng of Human Genomic DNA Male (Promega, catalog # G1471) was fragmented by using different enzymatic digestion times. After the adapter ligation was

completed, the single screen mode and double screen mode were used respectively (see Appendix II 1.2 of the user manual for details).

The library yield after double-sided selection is 15-25% loss compared with  single selection. But it is not recommended for low-quality DNA or input DNA is ≤50 ng.

• Sample quality difference: Whether the samples are degraded. For degraded samples, the enzymatic digestion time can be shortened in a gradient of 3-5 min according to the recommended time in the user manual.
• Sample solvent: Solvent contains EDTA or pH is too high (pH greater than 8.0, such as pH 8.5). When the sample solvent contains EDTA, if it is not purified, Enhancer Buffer is not added, or the solvent pH is too high, it will lead to insufficient fragmentation.

A. 50 ng blood gDNA samples containing EDTA (0-0.8 mM) were used for library preparation. The higher the EDTA concentration, the larger the enzymatic digestion fragment.

NadPrep EZ DNA Library Preparation Kitv2 is suitable for various types of samples, including whole blood gDNA, FFPE, etc. For the treatment of complex samples (such as FFPE samples), it is recommended to carry out quality control at the source. If there are impurities such as protein and phenols in DNA samples, the effect of enzymatic digestion may be affected.

• Sample solvent: It is recommended to use nuclease Free Water or 10 mM Tris-HCl (pH 7.5-8.0) to dissolve the DNA samples (the enzymatic fragmentation reagent is sensitive to the concentration of EDTA. Therefore, it is necessary to confirm the concentration of EDTA in the solvent in this step. If the solvent pH is too high, the enzyme activity might be affected, resulting in insufficient fragmentation (1.8X magnetic bead purification can be carried out if necessary).
• Sample quantification: It is recommended to use NanoDrop based on the principle of spectrophotometer as well as Qubit® and PicoGreen® based on double-stranded DNA fluorescent dye for quantitative analysis of general gDNA samples.
• Sample purity: High quality DNA input, with OD260/OD280=1.8-2.0；OD260/OD230=2.0-2.5.
• Sample fragment distribution: It is recommended to use 1% agarose gel electrophoresis for sample size detection or to use bioanalyzer (such as Agilent 2100) DIN value for sample integrity assessment. The samples can be simply classified into four grades as shown in the following figure (the basic four grade classification method; samples can also be further classified according to the actual situation). The enzymatic digestion time of different grades of FFPE samples can be adjusted according to the user manual.

Grading Standard as follows:

A:One clear  strip with about 15 kb in size

B+:One clear and slight trailing strip with about 15 kb in size

B:One indistinct strip with about 15 kb Consistent withg DNA One extra cycle more in size, with medium diffusion

C:Multiple strips ranging from 200 bp to 2,500 bp, with severe diffusion

D:Multiple strips ranging from 100 bp to 1 kp, with severe diffusion

No, Nuclease-Free water shall be added to  a total volume of 40μL. The composition of TE Solution is 10 mM Tris, 0.1 mM EDTA (pH 8.0), which can be used for library storage.

The degree of fragmentation of FERA Enzyme depends on time and temperature. The size of DNA fragment depends on the enzymatic digestion time, and the size of DNA fragment can be flexibly adjusted according to the enzymatic digestion time.