In silico digestion was performed using biopieces v0.48 (
Buffer 3
Manufactured by New England Biolabs
Sourced in China
Buffer 3.1 is a reagent designed for use in various laboratory applications. It functions as a buffering agent to maintain a specific pH range in experimental solutions. The core purpose of this product is to provide a stable and controlled chemical environment for diverse research procedures.
Automatically generated - may contain errors
Lab products found in correlation
Rnase inhibitor, by Takara Bio (2 mentions)
Lba cas12a, by New England Biolabs (2 mentions)
Gel extraction kit, by Qiagen (2 mentions)
Agilent 2100, by Agilent Technologies (2 mentions)
Bsiwi, by New England Biolabs (2 mentions)
E2 crimson template, by Takara Bio (2 mentions)
Bsiwi, by Takara Bio (2 mentions)
Engen sgrna synthesis kit, by New England Biolabs (2 mentions)
Plasmid plus midi kit, by Qiagen (2 mentions)
Quick ligation kit, by New England Biolabs (2 mentions)
Pxpr 050 vector, by Addgene (2 mentions)
Stbl3 chemically competent e coli cells, by Thermo Fisher Scientific (2 mentions)
Proteinase k, by New England Biolabs (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Quantstudio dx, by Thermo Fisher Scientific (2 mentions)
Proteinase k, by Thermo Fisher Scientific (2 mentions)
T4 dna ligase reaction buffer, by New England Biolabs (1 mentions)
Rnase, by Tiangen Biotech (1 mentions)
T4 dna polymerase, by New England Biolabs (1 mentions)
Esp3i, by Thermo Fisher Scientific (1 mentions)
24 protocols using buffer 3
1
In silico Genome Digestion Optimizes Plant DNA Analysis
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In silico digestion was performed using biopieces v0.48 (www.biopieces.org ) of the A. thaliana (TAIR 10), B. vulgaris ssp. vulgaris, B. rapa, O. sativa ssp. indica version 9311_BGF_2005, O. sativa ssp. japonica, and Z. mays (B73) nuclear reference genomes [38 (link), 45 (link)–48 (link)]. The per-base genome coverage by digestion fragments with a length between 150 and 420 bp was expressed relative to the nuclear reference sequence size. About two micrograms of genomic DNA were either digested with firstly MspI (60 U, 37 °C) and secondly ApeKI (15 U, 75 °C), or with MspI (60 U, 37 °C) followed by DpnII (30 U, 37 °C) added in two half units portions and in Buffer 3.1 (all obtained from NEB, MA) in a final volume of 60 μL for 20 h each, according to manufacturer’s instructions. Successful digestion was confirmed by gel electrophoresis. For plant-RRBS samples, a smear of fragments of different sizes and no evidence of non-digested, high-molecular mass molecules were observed, indicating successful digestion. Genomic DNA control samples taken along without restriction endonucleases showed a discrete high-molecular mass, indicating the absence of contaminating nucleases and persistent DNA quality despite the incubation procedure.
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In silico digestion was performed using biopieces v0.48 (
2
Cas9-Mediated DNA Repair and Ligation
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First, a 50-µl reaction mixture containing ~3 µg Cas9 protein, 10 µg sgRNA, and 3 µg DNA was prepared and incubated in buffer 3.1 (New England Biolabs) at 37°C overnight. The reaction was terminated by addition of 0.2 mg/ml RNase (Tiangen Biotech) and continued incubation at 37°C for 15 min. Then the reaction mixture was treated with SDS (to 1%), 1 mg/ml proteinase K, and 10 mM CaCl2 and incubated at 55°C for 30 min. Finally, the Cas9-digested DNA was recovered by ethanol precipitation.
T4 DNA polymerase (New England Biolabs) was used to repair the sticky end generated by 3′→5′ exonuclease activity of Cas9. A mixture of 3 µg Cas9-digested DNA, 100 µM deoxynucleoside triphosphates (dNTPs), 1× bovine serum albumin (BSA), and 0.5 µl T4 DNA polymerase was prepared in 1×T4 DNA ligase reaction buffer (New England Biolabs). The end repair mixture was then incubated at 12°C for 15 min, and reaction was terminated by incubation at 75°C for 20 min. Subsequently, end-repaired DNA was self-ligated or ligated with an additional DNA fragment in the ligation mixture, which contained 0.2×T4 DNA ligase reaction buffer (New England Biolabs), 15% (vol/vol) polyethylene glycol 4000 (PEG 4000), and 1 µl T4 DNA ligase (Thermo, Fisher Scientific), and then the mixture was incubated at 16°C overnight.
T4 DNA polymerase (New England Biolabs) was used to repair the sticky end generated by 3′→5′ exonuclease activity of Cas9. A mixture of 3 µg Cas9-digested DNA, 100 µM deoxynucleoside triphosphates (dNTPs), 1× bovine serum albumin (BSA), and 0.5 µl T4 DNA polymerase was prepared in 1×T4 DNA ligase reaction buffer (New England Biolabs). The end repair mixture was then incubated at 12°C for 15 min, and reaction was terminated by incubation at 75°C for 20 min. Subsequently, end-repaired DNA was self-ligated or ligated with an additional DNA fragment in the ligation mixture, which contained 0.2×T4 DNA ligase reaction buffer (New England Biolabs), 15% (vol/vol) polyethylene glycol 4000 (PEG 4000), and 1 µl T4 DNA ligase (Thermo, Fisher Scientific), and then the mixture was incubated at 16°C overnight.
3
Introducing Cohesive Ends for DNA Ligation
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PCR fragments that were to be ligated to bead-immobilised DNA, required cohesive ends. For the assembly set out in Figure 2C , a 5′-overhang in PCR product ‘frag1’ (Supplementary Table S4 ) was introduced by restriction with BspQI: a 30 μl reaction consisting of 150 pM DNA, 1× buffer 3.1 (NEB) and 30 units of BspQI (NEB), was incubated at 50°C for 2 h, followed by inactivation of the restriction enzyme by heating to 80°C for 20 min. 5′-Overhangs in fragT10 PCR fragments for the final fragment ligation in the ZIgE SpliMLiB library (Figure 3C , step viii) were introduced by restriction with Esp3I, in 50 μl reactions consisting of 70–100 pM of purified PCR fragment, 50 units of Esp3I (ThermoFisher Scientific), 1× buffer Tango (ThermoFisher Scientific) supplemented with 1 mM DTT. The restriction reactions were incubated at 37°C for 2 h followed by 20 min at 65°C to heat-inactivate Esp3I. In both cases, the restricted DNA was purified using the SPRI bead protocol described above.
4
SARS-CoV-2 Genome Detection Protocol
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All synthetic nucleic acid oligonucleotides of Tables S1–S4 diethylpyrocarbonate (DEPC)-treated water were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Lba Cas12a and buffer 3.1 were purchased from New England Biolabs (Beijing, China). RNase inhibitors were purchased from Takara Biotechnology Co., Ltd. (Dalian, China). The RNase-free environment throughout the experiments using DEPC-treated water and RNase-free tips and tubes. GX/P2V beta coronavirus was isolated by using Vero E6 cells.
5
Genotyping by Sequencing Protocol
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The double digest reactions were carried out in a volume of 25 μl containing ∼0.8 μg of genomic DNA, 5U of PstI and MseI, and 1× Buffer 3.1 (NEB). The reaction mixture was incubated at 37° for 2 hr and 65° for 30 min. Amplification and sequencing adapters with a unique barcode (6 bp or 8 bp) were ligated onto the digested DNA. Each sample was then amplified via PCR in a 50 μl reaction volume, containing 70-100 ng of adaptor-ligated DNA fragments and amplified with 22 cycles following the manufacturer’s protocol. Samples were electrophoresed on a 2% agarose gel, and DNA in the 300-450 bp size range (with indices and adaptors) was excised and purified using a Gel Extraction Kit (Qiagen). Each sample library was pooled in equal amounts and quantified using an Agilent 2100 bio analyzer (Agilent Technologies) and then paired-end 101 bp sequencing was performed using the Illumina HiSeq4000 platform at BGI (Shenzhen, China).
6
Cloning of E2-Crimson Template into lentiGuide-Puro
7
CRISPR Mutagenesis with Designed sgRNA
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The CRISPR mutagenesis protocol was based on Davis et al. [51 (link)]. Single guide RNA targeting genes of interest was designed using Benchling [Biology Software (2021) Retrieved from https://benchling.com ] based on Cas9 cutting efficiency [52 (link)]. The sgRNA template was synthesized as a primer (S1 Table, SWC4_sg132 or SWR1_sg1979) and served for sgRNA synthesis using the EnGen sgRNA Synthesis Kit (NEB, USA) following the manufacturer’s instructions. The sgRNA was then purified using the RNA Clean & Concentrator-25 Kit (Zymo) following the manufacturer’s instructions. RNA concentration and quality were assessed by NanoDrop spectrophotometer. For each CRISPR reaction: 2.5 μL concentrated sgRNA, 2 μL of EnGen Spy Cas9 NLS (NEB) in the presence of buffer 3.1 (NEB) in a final volume of 5 μL were incubated at 37°C for 30 min and kept on ice until use.
8
Quantitative LbCas12a Detection Assay
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LbCas12a detection was carried out in Buffer 3.1 (NEB), 50nM lbCas12a protein, 60nM of crRNA and 30nM labeled probe, and 5µL PCR product, and ddH2O to supplement the volume to 20µL. The reaction system was mixed and then incubated at 37 °C. Reactions proceeded for one hour on QuantStudio Dx (ABI) with fluorescent kinetics measured every 2 min. Each reaction was repeated in three biologically independent experiments.
9
Double-Digest Restriction Site-Associated DNA Sequencing
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The procedure was performed as described in previous studies (DaCosta and Sorenson 2014 (link)) with some modifications. First, the double-digest reactions were carried out in a volume of 25 μl containing ∼0.8 μg of genomic DNA, 5U of PstI and MseI, and 1× buffer 3.1 (NEB). The reaction mixture was incubated at 37 °C for 2 hr and 65 °C for 30 min Amplification and sequencing adapters with a unique barcode (5 or 6 bp) were ligated on to the digested DNA. Each sample was then amplified via PCR in a 50 μl reaction volume, containing 70–100 ng of adaptor-ligated DNA fragments, and amplified with 22 cycles following the manufacturer’s protocol. Samples were run on a 2% agarose gel, and DNA in the 300–450 bp size range (with indices and adaptors) was excised using a gel extraction kit (Qiagen). Each sample library was pooled in equal amounts and quantified using Agilent 2100 (Agilent Technologies) and real-time quantitative PCR, and then paired-end 101 bp sequencing was performed using the Illumina HiSeq4000 platform (BGI, Shenzhen, China).
10
CRISPR Plasmid Construction Protocol
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Forward and reverse-complement single-stranded oligonucleotide inserts containing 5′ BsmBI sites followed by single guide RNA sequences were purchased from IDT. Oligos were mixed at equimolar concentrations in NEB Buffer 3.1 and annealed using thermal cycler conditions listed in Supplemental Table S6 . Following annealing of complementary oligonucleotide inserts, inserts were ligated into BsmBI-digested pXPR_050 vector (Addgene cat. no. 96925) using the Quick Ligation Kit (NEB) according to manufacturer's protocol and the ligation product was transformed into Stbl3 chemically competent E. coli cells (Invitrogen) via heat shock. Sequence-confirmed clones were cultured and pDNA extracted and purified using the Plasmid Plus Midi Kit (Qiagen).
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