2-Ethynyl-adenosine (2-EA) and 2-Ethynyl-ATP (2-EATP) were purchased from Jena Bioscience (Jena, Germany). Azide-modified 2-methylnicotinic acid i midazolide (NAI-N3) from MedChem Express (Shanghai, China). Succinimidyl-[4-(psoralen-8-yloxy)]butyrate (SPB), DBCO-cy5 and azide-FAM were obtained from Sigma Aldrich. Trans-Cyclooctene-amine (TCO-amine) and methyltetrazine-NHS ester were obtained from Click Chemistry Tools (Scottsdale, USA). Tetrazine-cy3 (Tz-cy3) and azide-cy5 (N3-cy5) were purchased from Lumiprobe (Maryland, USA). All chemicals were used as received without further purification. The reactions were performed in RNase-free water and buffers. The oligonucleotides used in this work (Supplementary Table S1 ) were synthesized by Sangon Biological Co. Ltd (Shanghai, China). DNA marker, T4 DNA ligase, Exonuclease I, Exonuclease III, RiboLock RNase Inhibitor and Trizol Reagent were obtained from Takara Biotechnology Co. Ltd (Dalian, China). Poly(U) polymerase, phi29 DNA polymerase and shrimp alkaline phosphatase (rSAP) were purchased from New England Biolabs Ltd (Beijing, China) and RNase I from ThermoFisher Scientific.
Shrimp alkaline phosphatase rsap
Manufactured by New England Biolabs
Sourced in United States
Shrimp alkaline phosphatase (rSAP) is an enzyme used in molecular biology applications. It catalyzes the removal of 5' phosphate groups from DNA, RNA, and nucleotides.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by New England Biolabs (3 mentions)
Exonuclease 1, by New England Biolabs (2 mentions)
T4 polynucleotide kinase, by New England Biolabs (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Rnase 1, by Thermo Fisher Scientific (1 mentions)
Exonuclease 1, by Takara Bio (1 mentions)
Methyltetrazine nhs ester, by Vector Laboratories (1 mentions)
Dna marker, by Takara Bio (1 mentions)
Exonuclease 3, by Takara Bio (1 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Polyu polymerase, by New England Biolabs (1 mentions)
Phi29 dna polymerase, by New England Biolabs (1 mentions)
5 fluoroorotic acid 5 foa, by Thermo Fisher Scientific (1 mentions)
Gibson assembly cloning kit, by New England Biolabs (1 mentions)
Phusion dna polymerase, by New England Biolabs (1 mentions)
Zymolyase 20t, by MP Biomedicals (1 mentions)
Gene pulser xcell, by Bio-Rad (1 mentions)
Rosetta2 de3, by Addgene (1 mentions)
Hiscribe t7 quick high yield rna synthesis kit, by New England Biolabs (1 mentions)
13 protocols using shrimp alkaline phosphatase rsap
1
Covalent Labeling of Nucleic Acids
2
Molecular Cloning and Genetic Manipulation
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All chemicals were purchased from Sigma-Aldrich, unless otherwise stated. Phusion DNA polymerase, as well as all restriction endonucleases, T4 DNA ligase, shrimp alkaline phosphatase (rSAP) and Gibson Assembly cloning kit, were purchased from New England Biolabs. Zymolyase 20T was purchased from MP Biomedicals, 5-fluoroorotic acid (5-FOA) was purchased from Thermo Fisher Scientific. Primers for PCR amplification were purchased from Sigma-Aldrich (see ESI Table S3† for a list of oligonucleotides used in this study).
3
Infectious Begomovirus Partial Dimeric Clones
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Infectious begomovirus partial dimeric clones of Galium leaf distortion virus (GLDV) isolates (GLDV-1 and GLDV-2) were constructed as previously described (Bang et al., 2014 (link)). A fragment of 1.5 kb was released from monomeric constructs pG-GLDV-1 and pG-GLDV-2 by BamHI and SacI double digestion and cloned in a pGreen 0029 binary vector linearized with same enzymes to yield pG-GLDV-1 0.6mer and pG-GLDV-2 0.6mer constructs, respectively. GLDV-1 0.6mer and pG-GLDV-2 0.6mer intermediaries were linearized with BamHI and dephosphorylated with shrimp alkaline phosphatase (rSAP) (New England BioLabs) following the manufacturer’s instructions. Then, the fragments were ligated with a monomeric copy of a full-length viral genome released with BamHI from the corresponding monomeric construct to yield GLDV-1 1.6mer and pG-GLDV-2 1.6mer constructs. In each cloning step, positive clones were verified by enzymatic digestion. Positive partial dimeric clones were transformed into Agrobacterium tumefaciens GV3101 strain by electroporation (25 μF, 400 Ω, and 2,500 V) in a Gene PulserXCell (Bio-Rad) apparatus.
4
Single-Cell PCR Amplification and Sequencing
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PCR-reactions were run with 0.13 ng/µL to 0.33 ng/µL of single nucleus DNA as a template, 0.5 µM forward and reverse primers each (Supplementary file 13 and 0.2 mM dNTPs. For reactions performed with Phusion HF Polymerase, 1x Phusion HF Buffer and 0.02 U/µL Phusion HF Polymerase and for reactions with GoTaq Hot-Start Polymerase, 1x Green Buffer, 2.5 mM MgCl2 and 0.025 U/µL GoTaq Hot-Start Polymerase were used. The reactions were performed in a total volume of 15 µL or 30 µL. The amplification was run with a touchdown PCR program: 95°C/2 min - [95°C/30 s; T1/30 s (T1 declining by 0.5°C every cycle) -; 72°C/30 s]x10 - [95°C/30 s; T2/30 s; 72°C/30 s]x35–72°C/5 min. T1 and T2 are specific annealing temperatures for different primer combinations (see Supplementary file 9 ). PCR cleanup was performed with 2.9 U/µL Exonuclease I (New England Biolabs) and 0.14 U/µL Shrimp Alkaline Phosphatase (rSAP, New England Biolabs) at 37°C for 5 min and heat inactivated at 85°C for 10 min. Sanger sequencing reactions were performed at the Genomics Service Unit (LMU).
5
CRISPR-Cas9 RNP Complex Formation
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Escherichia coli strain Rosetta 2 DE3 carrying plasmid pMJ806 was obtained from Addgene. The Cas9 protein was prepared following the protocol as described (Anders and Jinek,2014 ). For in vitro transcription of guide RNA, pT7‐sgRNA derived gRNA‐specific construct was linearized at a unique restriction enzyme site NcoI, followed by dephosphorylation with Shrimp Alkaline Phosphatase (rSAP) (New England Biolabs, Ipswich, MA), and then purified with DNA Clean and Concentrator Kit (Zymo Research, Irvine, CA). sgRNA was synthesized by using the HiScribe T7 Quick High Yield RNA Synthesis Kit (New England Biolabs) following the manufacturer's instructions and purified with RNA Clean and Concentrator (Zymo Research, Irvine, CA). For each DNA/ribonucleoprotein reaction, 1 μg of sgRNA and 1 μg of Cas9 protein were mixed in Cas9 reaction buffer and incubated at room temperature for 15 min to form a ribonucleoprotein (RNP) complex. The resulting RNP was used for 2–3 reactions each with about 1 μg of PCR product in a total volume of 10 μL at 37 °C for 3 h. After inactivated at 65 °C for 15 min, the reactions were analysed with electrophoresis in 2% agarose gel.
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Escherichia coli strain Rosetta 2 DE3 carrying plasmid pMJ806 was obtained from Addgene. The Cas9 protein was prepared following the protocol as described (Anders and Jinek,
6
TeSLA Telomere Length Measurement Protocol
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Genomic DNA was extracted using the Gentra Puregene DNA Extraction Kit (Qiagen) according to the manufacturer's instructions and quantified on a NanoDrop (Thermo Scientific). TeSLA measurements were performed as previously described (Lai et al, 2017 ). In brief, T4 DNA ligase (New England Biolabs), 1 mM ATP, 10 − 3 μM of TeSLA‐Ts, and 50 ng of isolated genomic DNA were mixed in 1× CutSmart buffer (New England Biolabs) and incubated at 35°C for 12–16 h. The mixture was then digested with CviAII, BfaI, NdeI, and MseI (New England Biolabs) to generate DNA fragments with 5′ AT and TA overhangs. Shrimp alkaline phosphatase (rSAP; New England Biolabs) was added to the digested mixture to remove 5′ phosphate from each DNA fragment. The mixture was combined with T4 DNA ligase, 1 mM ATP, 1 μM of AT adapter, and 1 μM of TA adapter in 1× CutSmart buffer to incubate at 16°C for 12–16 h. Multiple PCRs were then performed using FailSafe Enzyme Mix (Lucigen) with 1× FailSafe buffer H containing 0.25 μM AP/TeSLA‐TP primers and 40 pg of ligated DNA. PCR products were resolved on a 0.85% agarose gel (1.5 V/cm for 19 h). After gel electrophoresis, Southern blot is applied to detect amplified telomeres.
7
Cloning and Expression of GFP
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The CloneJET PCR Cloning Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to obtain intermediate constructs by direct PCR and restriction/ligation cloning of required genetic elements. The pRSET-EmGFP plasmid (Thermo Fisher Scientific) was used as an expression vector containing the GFP gene. High-fidelity restriction endonucleases BamHI-HF, BglII-HF, EcoRI-HF, NcoI-HF, and PstI-HF; T4-DNA-ligase; T4-polynucleotide-kinase; and shrimp alkaline phosphatase (rSAP) were purchased from New England Biolabs (Ipswich, MA, USA). The oligonucleotides were synthesised by the solid-phase method and purified by preparative polyacrylamide gel electrophoresis (PAGE) by Syntol LLC (Russia). Q5® High-Fidelity DNA Polymerase (New England Biolabs) was used for all polymerase chain reaction (PCR). Ultrafree-DA Centrifugal Filter Units (Merck, Kenilworth, NJ, USA) were used for DNA extraction from agarose gel. The ZymoPURE™ Plasmid Miniprep Kit (Zymo Research, Irvine, CA, USA) was used for plasmid DNA purification. The authenticity of the plasmids was confirmed by Sanger sequencing performed by Eurogen CJSC (Russia). E. coli strain NiCo21(DE3) (#C2529H, New England Biolabs, Ipswich, MA, USA) was used for cloning and expression experiments according to the manufacturer’s protocol.
8
Purification and E2 Charging Assay for Ubiquitin Enzymes
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Human N-terminal His-tagged ubiquitin-like modifier activating enzyme 1 (UBA1) was expressed in E. coli BL21 RIL and purified via IMAC and size exclusion chromatography53 (link). The C-terminally His-tagged E2 enzyme UbcH7 was expressed in E. coli BL21 (DE3) and purified via IMAC54 (link). Ub was expressed in E. coli BL21 (DE3) and purified via IMAC and cation exchange chromatography7 (link).
The E2 charging assay was performed with 250 nM UBA1, 4 µM UbcH7, 15 µM Ub, 25 mM Tris·HCl (pH 7.5), 50 mM NaCl, 0.1 mM DTT, 10 mM MgCl2, and started with 0.2 mM of the indicated nucleotide (ATP, Ap3A, or Ap4A). For the SAP control, the nucleotides were pre-incubated with 0.5 units of shrimp alkaline phosphatase (rSAP) (New England Biolabs) at 37 °C for 30 min, followed by inactivation of the rSAP at 65 °C for 15 min. The reaction mixtures were incubated at 20 °C and enzymatic reactions were stopped at the indicated time points by the addition of 6× non-reducing SDS loading dye. To the DTT controls 1 µL of 1 M DTT was added before stopping the reaction. Samples were separated by 15% SDS-PAGE gels and stained with Coomassie blue.
The E2 charging assay was performed with 250 nM UBA1, 4 µM UbcH7, 15 µM Ub, 25 mM Tris·HCl (pH 7.5), 50 mM NaCl, 0.1 mM DTT, 10 mM MgCl2, and started with 0.2 mM of the indicated nucleotide (ATP, Ap3A, or Ap4A). For the SAP control, the nucleotides were pre-incubated with 0.5 units of shrimp alkaline phosphatase (rSAP) (New England Biolabs) at 37 °C for 30 min, followed by inactivation of the rSAP at 65 °C for 15 min. The reaction mixtures were incubated at 20 °C and enzymatic reactions were stopped at the indicated time points by the addition of 6× non-reducing SDS loading dye. To the DTT controls 1 µL of 1 M DTT was added before stopping the reaction. Samples were separated by 15% SDS-PAGE gels and stained with Coomassie blue.
9
Dephosphorylation of RNA Oligos
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Dephosphorylation assays were performed according to the manufacturer's protocol either with Shrimp Alkaline Phosphatase rSAP or RNA 5′ Pyrophosphohydrolase RppH (New England Biolabs) to remove all phosphates or to obtain monophosphorylated RNA oligos, respectively.
10
In Vitro Transcription and mRNA Purification
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The eluted double-stranded cDNAs (6 μl) were combined with IVT mix [2 μl each of A/G/C/UTP solution, 4 μl of 10× T7 reaction buffer, and 2 μl of T7 enzyme from the TranscriptAid T7 High Yield Transcription Kit (Invitrogen, K0441)] and incubated for 13 h at 37 °C. 3 μl of Exonuclease I (New England Biolabs, M0293L) and 3 μl of Shrimp Alkaline Phosphatase (rSAP) (New England Biolabs, M0371L) were added and incubated for 15 min at 37 °C, afterward, 2.6 μl of fragmentation buffer (Invitrogen, AM8740) was added and incubated for 15 min at 70 °C. After adding 2.86 μl of stop buffer, the aRNAs were purified using 3× Speedbead magnetic carboxylate (Cytiva, 17357672) and eluted with 7 μl of 10 mM Tris-HCl, pH 8.0.
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