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Authors: Piyush Jain, Long T Nguyen, Santosh Rananaware
Group: Jain Lab
Summary: The CRISPR-Enhance SARS-CoV-2 detection kit has been designed to detect fragments of the Nucleocapsid (“N”) gene and Envelope gene (E) of SARS-CoV-2. An included third target is the human RNase P POP7 gene (“RP”) which serves as a control for the extraction of the clinical sample in the absence of a positive SARS-CoV-2 result.The entire reaction from RT-LAMP amplification to CRISPR-based detection of the target analytes can be performed in approximately one hour.The CRISPR-Enhance kit comprises of two steps. Step one is a reverse transcriptase loop-mediated amplification (RT-LAMP) where targeted SARS-CoV-2 genomic RNA is reverse transcribed to DNA, and this DNA is amplified by a strand-displacing DNA polymerase. Step two is the transcription of the amplified DNA to activate the collateral cleavage activity of a CRISPR complex programmed to the target RNA sequence. This collateral activity results in cleavage of nucleic acid reporters, resulting in a visual readout using a paper strip in appoximately 2 mins.
Proper citation: Piyush Jain, Long T Nguyen, Santosh Rananaware 2020. CRISPR-Enhance Lateral Flow Assay. protocols.io dx.doi.org/10.17504/protocols.io.bk5zky76 Copy
Authors: J Jobu Babin, Haritima Chauhan, Feng Liu
Summary: AbstractLying about personal qualifications for a job or in college admissions is common and has recently become a high profile issue. In this paper, we explore the decision to misrepresent one’s self and subsequently determine whether honesty oaths impact truthful reporting of a personal characteristic. To do this, we execute a field experiment on Amazon MTurk using a self-reporting task involving worker eye color. We provide empirical evidence that honesty oaths elicit more truthful behavior, primarily impacting “extreme” lies. Oath-taking decreased the cost associated with misreporting personal characteristics by 70%. Oaths marginally decrease shirking.
Proper citation: J Jobu Babin, Haritima Chauhan, Feng Liu 2020. Supplemental Resources for You Can’t Hide Your Lying Eyes: Honesty Oaths and Misrepresentation. protocols.io dx.doi.org/10.17504/protocols.io.bix3kfqn Copy
Authors: Courtney Comrie
Summary: This protocol will provide a basic guide creating Tensor maps.Note: Steps may vary based upon data.
Proper citation: Courtney Comrie 2020. Creating Tensor Maps. protocols.io dx.doi.org/10.17504/protocols.io.8jyhupw Copy
Authors: Kizhakke Mattada Sathyan, Thomas G. Scott, Michael J. Guertin
Summary: The next step in developing the ARF-AID system is to tag the gene of interest with AID. The ARF-AID system requires full-length AID (Figure 1) because the characterized interaction domains of AID with ARF are domains III and IV. Domains I and II are involved in the interaction with TIR1. The mini-AID lacks domains III and IV and will not interact with ARF to stabilize the protein in the absence of auxin (Sathyan et al., 2019). Note that the antibiotic selection marker (HygroR) is co-transcribed with AID and the protein products are separated during translation. Therefore, the resistance marker will be expressed at levels comparable to the target protein.
Proper citation: Kizhakke Mattada Sathyan, Thomas G. Scott, Michael J. Guertin 2020. Basic Protocol 2: Tagging a gene of interest with AID. protocols.io dx.doi.org/10.17504/protocols.io.bdyei7te Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: The NEBExpress MBP Fusion and Purification System takes advantage of the strong Ptac promoter and the translation initiation signals of maltose binding protein (MBP) to enhance solubility and expression levels of a desired protein inE. coli. The resulting product is an MBP fusion protein, which is then purified by affinity chromatography.ReliableE. coli expression: substantial yields (up to 100 mg/L)Fusion to MBP has been shown to enhance the solubility of proteins expressed inE. coli(1)Two-step purification: amylose elution followed by TEV Protease cleavage and Ni resin isolation results in a highly pure tag-free target proteinGentle elution with maltose; no detergents or harsh denaturants required
Proper citation: New England Biolabs 2020. NEBExpress MBP Fusion and Purification System (NEB #E8201). protocols.io dx.doi.org/10.17504/protocols.io.bfayjifw Copy
Authors: Matthew Sullivan
Group: VERVE Net, Sullivan Lab
Proper citation: Matthew Sullivan 2016. 16S Universal Bacterial PCR. protocols.io dx.doi.org/10.17504/protocols.io.dfr3m5 Copy
Authors: Victoria Linderberg
Group: Thomas Crouzier Lab
Summary: This protocol describes how to wash QCM-D sensors before usage and how to modify their surface using a composition of symmetric (poly)ethylene glycol (PEG) thiols consisting of 99% dS-PEG and 1% dS-PEG-biotin.
Proper citation: Victoria Linderberg 2016. Protocol for washing and modifying QCM-D sensors. protocols.io dx.doi.org/10.17504/protocols.io.ex6bfre Copy
Authors: HANNIGAN GC, GRICE EA, ET AL
Group: VERVE Net, Club Grice
Summary: This protocol provides a method for predicting the locations of the open reading frames (ORFs) using the Glimmer3 toolkit. Methods based on the publication: Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." mBio 6.5 (2015): e01578-15.
Proper citation: HANNIGAN GC, GRICE EA, ET AL 2016. Script P3: Open Reading Frame Prediction. protocols.io dx.doi.org/10.17504/protocols.io.efpbbmn Copy
Authors: Bioline
Group: Bioline
Summary: Protocol for RNA Purification form Plasma or Serum, using the ISOLATE II Biofluids RNA Kit. This protocol includes the lysate preparation procedure.
Proper citation: Bioline 2016. Total RNA Purification from Plasma or Serum (ISOLATE II Biofluids RNA Kit). protocols.io dx.doi.org/10.17504/protocols.io.f5hbq36 Copy
Authors: Angela Piersanti
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Angela Piersanti 2019. Transfection by Electroporation in Euplotes crassus. protocols.io dx.doi.org/10.17504/protocols.io.2a9gah6 Copy
Authors: Payal Patel, Bethany Rozeboom, Tarah-Anne Abrigo
Proper citation: Payal Patel, Bethany Rozeboom, Tarah-Anne Abrigo 2020. Western Blot . protocols.io dx.doi.org/10.17504/protocols.io.bfiijkce Copy
Authors: Cristian Riccio
Summary: If the RNA you have extracted is not pure and contains some residual contamination, as shown by poor Nanodrop ratios, you can reprecipitate the RNA, wash it and re-dissolve it to purify it.
Proper citation: Cristian Riccio 2019. RNA re-precipitation protocol. protocols.io dx.doi.org/10.17504/protocols.io.3signce Copy
Authors: Michelle Daniel, Marda Jorgensen
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Proper citation: Michelle Daniel, Marda Jorgensen 2020. Imaging Mass Cytometry Modality Overview . protocols.io dx.doi.org/10.17504/protocols.io.bgatjsen Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This protocols is for PCR using Q5® High-Fidelity DNA Polymerase (M0491)
Proper citation: New England Biolabs 2015. PCR Using Q5® Hot Start High-Fidelity DNA Polymerase (M0493). protocols.io dx.doi.org/10.17504/protocols.io.cjtunm Copy
Authors: George Laliotis
Proper citation: George Laliotis 2020. Effective Identification of Protein-Protein Interaction using RIME-IP. protocols.io dx.doi.org/10.17504/protocols.io.bqx8mxrw Copy
Authors: Mathias Middelboe, Amy M. Chan, and Sif K. Bertelsen
Group: VERVE Net, Suttle Laboratory of Marine Molecular Microbiology and Virology
Summary: Described is the procedure used to perform a onestep growth curve for cyanophage BBC1-P1 via plaque assay on Synechococcus sp. BBC1 (Suttle and Chan 1993).
Proper citation: Mathias Middelboe, Amy M. Chan, and Sif K. Bertelsen 2016. One-step growth experiments (cyanophages). protocols.io dx.doi.org/10.17504/protocols.io.dra52d Copy
Authors: Kristoffer Bach Falkenberg, Cristina Hernandez Rollan, Maja Rennig, Andreas Birk Bertelsen, Morten Norholm
Summary: Lytic polysaccharide monooxygenses (LPMOs) are enzymes that play a critical role in breaking the chemical bonds of the most abundant polymers found in recalcitrant biomass, such as cellulose and chitin. LyGo cloning (Lytic Polysaccharide Monooxygenase Golden Gate cloning) is a versatile heterologous expression platform for LPMOs, which is compatible with cloning both PCR products and synthetic gene fragments with a simple 15-minute assembly step. The method allows for parallel construction of multiple expression vectors, enabling exploration of several expression strategies. The open-source LyGo collection consists of vectors for some of the most relevant model organisms used for protein production in both academic and industrial settings.This protocol describes how to clone LyGo fragments into LyGo vectors.
Proper citation: Kristoffer Bach Falkenberg, Cristina Hernandez Rollan, Maja Rennig, Andreas Birk Bertelsen, Morten Norholm 2020. LyGo cloning. protocols.io dx.doi.org/10.17504/protocols.io.bdmqi45w Copy
Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: LDL and VLDL are separated from HDL using a precipitation reagent. Then the HDL fraction is measured for either TC or TG using the same reagents for total cholesterol or triglyceride.
Proper citation: Peter Havel 2019. UC Davis - HDL Protocol. protocols.io dx.doi.org/10.17504/protocols.io.ynsfvee Copy
Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary:If your target cells are the labeled cells (the positive fraction), use the Streptavidin Nanobeads Protocol – Positive Selection. If your target cells are the unlabeled cells (negative fraction), use the Streptavidin Nanobeads Protocol - Negative Selection.
Target cells are isolated by incubating your sample with a Biotin-conjugated antibody or antibody cocktail, followed by incubation with magnetic Streptavidin Nanobeads (Cat. No. 480015/480016). The magnetically labeled fraction is retained by the use of a magnetic separator. The untouched cells are collected. These are the cells of interest; do not discard the liquid. Some of the downstream applications include functional assays, gene expression, phenotypic characterization, etc.Note: This procedure is optimized for the isolation of 107 to 2 x 108 cells per tube. If working with fewer than 107 cells, keep volumes as indicated for 107 cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water.
Scale up volumes if using 14 mL tubes and Magnet, and place the tube in the magnet for 10 minutes.
Proper citation: Sam Li 2019. MojoSort™ Streptavidin Nanobeads Protocol - Positive Selection. protocols.io dx.doi.org/10.17504/protocols.io.7wghpbw Copy
Authors: Liu Liu
Summary: This protocol is for consistently applying fluorescent dyes to Neuropixels probes and other types of silicon probes, which allows imaging and reconstruction of the electrode tracks in the brain in post hoc histology.
Proper citation: Liu Liu 2019. Painting Neuropixels probes and other silicon probes for electrophysiological recordings. protocols.io dx.doi.org/10.17504/protocols.io.wxqffmw Copy
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