Nanodrop uv vis spectrometer

Aβ₄₀ (Invitrogen, KHB3481) and Aβ₄₂ (Invitrogen, KHB3441) specific ELISA assay kit was used to quantify the concentration of two isoforms of β-amyloid secreted by neurons. 25 μl of neuronal culture medium was used for each well and the assay was performed following the manufacturer’s protocol. We used a global DNA methylation LINE-1 kit (Active Motif, 55,017) to quantify global DNA methylation level. Genomic DNA (gDNA) was extracted using a PureLink Genomic DNA Mini Kit (Invitrogen, K182001); and concentration was determined via NanoDrop UV-Vis spectrometer (ThermoFisher). 100 μg gDNA were applied to each well to determine their methylation level following the manufacturer’s protocol. All absorbance measurements were performed using a SpectraMax iD3 plate reader (Molecular Device).

The DNA oligonucleotide used in this study is the self-complementary sequence 5′-CGC AAA TTT GCG-3′, purchased from Integrated DNA Technologies (IDT) at desalt-grade purity and further purified by dialysis using membrane tubing with a 0.5 kD MWCO (Spectrum Micro Float-A-Lyzer) and distilled water at 0 °C for 8 h. Hoechst 33258 (H) was purchased from Chemodex and is used as received. The duplex DNA bound with Hoechst 33258 is denoted by H-DNA. To avoid the formation of DNA hairpins, all DNA samples were prepared in pH 7.4 deuterated buffer with 5 mM sodium phosphate and 200 mM sodium chloride. pH values were adjusted with 1 M HCl and NaOH solutions and then checked with pH meter (Fisherbrand accumet AB150). The concentration of oligonucleotide and H was confirmed on a NanoDrop UV/vis spectrometer (Thermo Scientific). H-DNA complexes were prepared by mixing buffer solutions in a 1:1 dsDNA-to-H mole ratio. We use C_D to refer to the total concentration of native and H-DNA (the equivalent value of dsDNA). Prior to measurements, all samples were annealed by heating to 95 °C for 3 min and then cooling gradually to room temperature over 10–15 min. For IR measurements, labile protons of DNA and H were HD exchanged in deuterated water (D₂O, Cambridge Isotopes) and lyophilized before dissolving into the deuterated buffer.

For each CLL sample two multiplex long-PCRs were performed with the two primer pools using PrimeSTAR GXL DNA Polymerase (Takara Bio Inc.), 70 ng of genomic DNA, in a final volume of 25 uL. Thermal cycling conditions were 98 °C for 10 minutes, 60 °C for 15 seconds, 68 °C for 4 minutes (35 cycles) and 12 °C hold for both the two long-PCR.
Since 3 amplicons of pool 1 had a very similar size of about 1.3 kb and were not easily distinguishable by 1% agarose gel electrophoresis, a restriction enzyme of these critical amplicons was made, and the BglII restriction enzyme (10,000 units/mL, New England BioLabs Inc.) was finally selected to verify their successful amplification and discriminate them. In detail, 5 uL of the primer pool 1 PCR products were incubated with 0.5 units of BglII and 1uL of NEBuffer 3.1 in 1 hour at 37 °C. Digestion products were visualized by SYBR Safe on agarose gel 1%.
PCR products from both the long PCR were purified with the QIAquick PCR Purification Kit (Qiagen) in an elution volume of 30 uL, and the DNA concentration and purity was measured with a Qubit 2.0 fluorometer (Life Technologies) and Nanodrop UV-Vis spectrometer (Thermo Fisher Scientific).
Two uL of pool 1 and pool 2 purified amplicons were visualized by SYBR Safe on agarose gel 1.0% (Supplementary Fig. S1).

Protein, mRNA, and DNA were simultaneously isolated from dissected mouse tissue using the AllPrep DNA/RNA/Protein Mini Kit (QIAGEN, 80004) following the manufacturer's provided protocol. The concentrations of RNA and DNA were determined by measuring the absorbance at 260 and 280 nm using a Nanodrop UV–Vis Spectrometer (ThermoFisher). The protein precipitates were resuspended and fully homogenized in 250 µl of RIPA buffer containing 0.5% SDS by a high intensity ultrasonic water bath at 50A for 30 s (QSonica 500 with a cup horn). The lysates were subsequently normalized for protein content using the BCA protein assay (ThermoFisher).

Protein extraction was performed as previously described [6 (link)]. In brief, Jurkat cells were lysed with 50 mM phosphate buffer (pH 7.4) containing the Roche Complete Mini protease inhibitor cocktail and then homogenized on ice with a microprobe sonicator until the turbid mixture turned nearly clear with no visible cells left. The homogenate was centrifuged at 10,000 g at 4 °C for 20 min, and the total protein extract in the supernatant was collected. Protein concentration was measured by absorbance at 280 nm using a NanoDrop UV-Vis spectrometer (Thermo Fisher).

Protein extraction was performed as previously described [4 (link)]. In brief, HS-Sultan cells were lysed with 50 mM phosphate buffer (pH 7.4) containing the Roche Complete Mini protease inhibitor cocktail and then homogenized on ice with a microprobe sonicator until the turbid mixture turned nearly clear with no visible cells left. The homogenate was centrifuged at 10,000 g at 4 °C for 20 min, and the total protein extract in the supernatant was collected. Protein concentration was measured by absorbance at 280 nm using a NanoDrop UV-Vis spectrometer (Thermo Fisher).