The on-plate ATAC-seq was modified based on the original ATAC-seq method [23 (link)] with the following modifications. First, 50,000 keratinocytes were seeded on a 96-well plate (Breiner Bio-one, Monroe, North Carolina, USA, Cat# 675180). After inducing differentiation for 4 days, the cells were washed briefly with phosphate-buffered saline (PBS) two times, and permeabilized with lysis buffer (10 mM Tris-Cl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.05 % NP-40) for 10 min. After this permeabilization step, the supernatant were removed, and replaced with 50 μL transposase and buffer mix (25 μL TD (2x reaction buffer from Nextera kit, Illumina, San Diego, California, USA), 5 μL TDE1(Tn5 Transposase from Nextera kit, Illumina), 20 μL nuclease-free H2O). After 30 min of incubation at 37 °C, a total volume of 250 μL Buffer PB (Qiagen, Hilden, Germany) was mixed with the reaction before DNA purification and polymerase chain reaction (PCR) enrichment. Each replicate of ATAC-seq data used in this study was generated individually using 50,000 keratinocytes growing in each well of a 96-well plate under differentiation conditions.
Pb buffer
Manufactured by Qiagen
Sourced in Germany, United States
PB buffer is a laboratory reagent used in nucleic acid purification protocols. It serves as a binding buffer that facilitates the adsorption of nucleic acids to a solid support, such as a silica-based matrix, during the purification process. The core function of PB buffer is to create the appropriate conditions for the efficient capture and retention of nucleic acids, enabling their subsequent washing and elution.
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Lab products found in correlation
Pe buffer, by Qiagen (6 mentions)
Tagment dna buffer, by Illumina (4 mentions)
Tagment dna enzyme, by Illumina (4 mentions)
Agencourt ampure xp, by Beckman Coulter (4 mentions)
Cutsmart buffer, by New England Biolabs (3 mentions)
Pcr purification kit, by Qiagen (3 mentions)
Miseq high throughput dna sequencer, by Illumina (2 mentions)
Veraseq ultra, by Qiagen (2 mentions)
Qiaprep spin column, by Qiagen (2 mentions)
Klenow exo, by New England Biolabs (2 mentions)
Nebuffer 2, by New England Biolabs (2 mentions)
Tbe urea gel, by Bio-Rad (2 mentions)
Amersham typhoon imager, by GE Healthcare (2 mentions)
User enzyme, by New England Biolabs (2 mentions)
Qiaquick 96 pcr purification kit, by Qiagen (2 mentions)
Zymo viral dna rna buffer, by Qiagen (2 mentions)
Minelute pcr purification kit, by Qiagen (2 mentions)
Plasmid plus midi kit, by Qiagen (2 mentions)
Electrocompetent 10b cells, by New England Biolabs (2 mentions)
4d nucleofector system, by Lonza (2 mentions)
35 protocols using pb buffer
1
High-throughput ATAC-seq in Keratinocytes
2
Optimized Ancient DNA Sequencing Protocol
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Tooth powder was obtained in dedicated clean rooms at the University of Tennessee, Knoxville using a freezer mill for 18 individuals and at Harvard Medical School by drilling for 2 individuals. DNA extraction for all samples was performed using a method optimized to retain small DNA fragments either manually47 (link),48 (link) or with an automated liquid handler using silica-coated magnetic beads49 (link). We prepared double-stranded Illumina sequencing libraries, pretreating with the enzyme uracil-DNA glycosylase (UDG) to minimize analytical artifacts due to the characteristic cytosine-to-thymine errors in ancient DNA25 , using an automated liquid handler and substituting the MinElute columns used for cleaning up reactions with silica-coated magnetic beads and buffer PB (Qiagen), and the MinElute column-based PCR cleanup at the end of library preparation with SPRI beads50 (link),51 (link). We enriched the libraries for sequences that overlapped both mtDNA52 (link) and about 1.24 million nuclear targets for two rounds of enrichment38 (link),53 (link),54 (link), either independently (1240k and MT separately) or together (1240kplus). We sequenced the enriched products on an Illumina NextSeq500 using v.2 150 cycle kits for 2 × 76 cycles and 2 × 7 cycles to read the indices. Skeletal material from all 20 ancient individuals screened for this project yielded usable DNA data.
3
CRISPR-Cas9 Editing Substrate Preparation
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The oligonucleotides listed in the Supplementary Sequences were obtained from IDT. Complementary sequences were combined (5 μL of a 100 μM solution) in Tris buffer and annealed by heating to 95 °C for 5 min, followed by a gradual cooling to 45 °C at a rate of 0.1 °C/s to generate 60-bp dsDNA substrates. Purified fusion protein (20 μL of 1.9 μM in activity buffer) was combined with 1 equivalent of appropriate sgRNA and incubated at ambient temperature for 5 min. The 60-mer dsDNA substrate was added to final concentration of 125 nM and the resulting solution was incubated at 37 °C for 2 h. The dsDNA was separated from the fusion by the addition of Buffer PB (100 μL, Qiagen) and isopropanol (25 μL) and purified on a EconoSpin micro spin column (Epoch Life Science), eluting with 20 μL of Tris buffer. The resulting edited DNA (1 μL was used as a template) was amplified by PCR using the HTS primer pairs specified in the Supplementary Sequences and VeraSeq Ultra (Enzymatics) according to the manufacturer's instructions with 13 cycles of amplification. PCR reaction products were purified using RapidTips (Diffinity Genomics), and the purified DNA was amplified by PCR with primers containing sequencing adapters, purified, and sequenced on a MiSeq high-throughput DNA sequencer (Illumina) as previously described.32
4
Optimizing LAMP and qPCR Reactions
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For studying kit buffer inhibitors, LAMP and qPCR reactions were spiked to 5 × 104 cp/rxn λ phage DNA (NEB) and supplemented with half-log dilutions of either Koptec 200-proof ethanol (V1001, Decon Labs, King of Prussia, PA, USA), Viral RNA Wash Buffer 1x (R1034-2-48, Zymo Research, Tustin, CA, USA), Buffer PE (19065, Qiagen, Germantown, MD, USA), Zymo DNA/RNA Shield 1x (R1200-125), Zymo Viral DNA/RNA Buffer (D7020-1-100), or Qiagen Buffer PB (19066) to the appropriate final concentration. For selecting the optimal TPW, LAMP and qPCR reactions were spiked with 1 µL of 5 × 104 cp/µL λ phage DNA, diluted to 10 µL, and an additional 1 µL was added of either nuclease-free water, 200 proof ethanol, isopropanol (BP2618-500, Thermo Fisher Scientific, Waltham, MA, USA), 1-butanol (3000-04, Mallinckrodt Chemicals), isopentanol (2992-04, Mallinckrodt Chemicals), 1-hexanol (H13303–100 mL, MilliporeSigma, St. Louis, MO, USA), 1-heptanol (H2805-250 mL, MilliporeSigma), 1-octanol (SHBH2844V, MilliporeSigma), 1-nonanol (131210–100 mL, MilliporeSigma), 1-decanol (2397563–50 g, MilliporeSigma), 1-undecanol (MKCG3271, MilliporeSigma), 2-dodecanol (D221503-5G, MilliporeSigma), 5 cSt silicone oil (317667-250 mL, MilliporeSigma), or Fluorinert FC-40 (ZF-0002-1308-0, 3 M, St. Paul, MN, USA).
5
One-Pot DNA Cleavage Purification
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Following one-pot DNA cleavage, the 50 µL reaction mixture was transferred into a 1.7 mL centrifuge tube. Next, 250 µL of buffer PB (Qiagen) was added to the mixture and after complete mixing, the solution was transferred into a Zymo-Spin I column (Zymo Research, CA) and centrifuged at 21,000 × g for 30 sec. The centrifuge flow-through was discarded and 400 µL of buffer PE (Qiagen) was added to the column. After centrifugation and discarding the flow-though, the PE buffer wash step was repeated one more time. The column was then transferred into a clean 1.7 mL centrifuge tube and 10 µL of ddH2O was directly added to the column matrix. To ensure maximum elution of cleavage products, the column was placed at room temperature for at least 5 min. Next, the column was centrifuged to acquire the purified cleavage products.
6
P1 Nuclease Assay for Plasmid DnaA Binding
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For the P1 nuclease assay, 100 ng pAP205 plasmid was incubated with increasing concentrations of DnaA-6xHis (0.14, 0.54, 1, and 6.3 μM), when required, in P1 buffer (25 mM Hepes-KOH (pH 7.6), 12% (v/v) glycerol, 1 mM CaCl2, 0.2 mM EDTA, 5 mM ATP, 0.1 mg/ml BSA), at 30°C for 12 min. 0.75 unit of P1 nuclease (Sigma), resuspended in 0.01 M sodium acetate (pH 7.6) was added to the reaction and incubated at 30°C for 5 min. 220 μl of buffer PB (Qiagen) was added and the fragments purified with the minElute PCR Purification Kit (Qiagen), according to manufacturer’s instructions. Digestion with BglII, NotI or ScaI (NEB) of the purified fragments was performed according to manufacturer’s instructions for 1 h at 37°C. Digested samples were resolved on 1% agarose gels in 0.5xTAE (40 mM Tris, 20 mM CH-COOH, 1 mM EDTA PH 8.0) and stained with 0.01 mg/mL ethidium bromide solution afterward. Visualization of the gels was performed on the Alliance Q9 Advanced machine (Uvitec). Images were processed in CorelDraw X7 software. For all experiments at least three independent replicates were performed with various concentrations of DnaA. To quantify the results, background-corrected band intensities were determined using ImageJ, values were normalized against the total signal in a lane in MS Excel, and plotted using GraphPad.
7
Preparation of Cy3-Labeled dsDNA Substrates
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Sequences of 80-nt unlabeled strands are listed in the Supplementary Sequences and were ordered as PAGE-purified oligonucleotides from IDT. The 25-nt Cy3-labeled primer listed in the Supplementary Sequences is complementary to the 3′ end of each 80-nt substrate. This primer was ordered as an HPLC-purified oligonucleotide from IDT. To generate the Cy3-labeled dsDNA substrates, the 80-nt strands (5 μL of a 100 μM solution) were combined with the Cy3-labeled primer (5 μL of a 100 μM solution) in NEBuffer 2 (38.25 μL of a 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT, pH 7.9 solution, New England Biolabs) with dNTPs (0.75 μL of a 100 mM solution) and heated to 95 °C for 5 min, followed by a gradual cooling to 45 °C at a rate of 0.1 °C/s. After this annealing period, Klenow exo– (5 U, New England Biolabs) was added and the reaction was incubated at 37 °C for 1 h. The solution was diluted with Buffer PB (250 μL, Qiagen) and isopropanol (50 μL) and purified on a QIAprep spin column (Qiagen), eluting with 50 μL of Tris buffer.
8
CRISPR-Cas9 Editing Protocol
9
Preparation of Cy3-Labeled dsDNA Substrates
10
PAM Identification Assay for Cas12a Orthologs
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