Protocols   Select Another Resource Report Type

Authors: David Eccles
Summary: This protocol is for comparing different samples at the transcript level, using long reads that are mapped to transcripts.Input(s): demultiplexed and oriented fastq files (see protocol Preparing Reads for Stranded Mapping), transcript reference fasta file, annotation fileOutput(s): transcript table, sorted by differential coverage, annotated with gene name / description / location

Proper citation: David Eccles 2019. Stranded Transcript Count Table Generation from Long Reads. protocols.io https://dx.doi.org/10.17504/protocols.io.594g98w Copy   

•   Source:

Authors: David Dilworth

Proper citation: David Dilworth 2018. A General Protocol for Western Blotting Mammalian Cell Lysates. protocols.io https://dx.doi.org/10.17504/protocols.io.pxndpme Copy   

•   Source:

Authors: Bao Thai
Group: Stephen Floor Lab

Proper citation: Bao Thai 2018. Western Lysis Buffer (WLB) 250 mL. protocols.io https://dx.doi.org/10.17504/protocols.io.mn7c5hn Copy   

•   Source:

Authors: Angel Justiz-Vaillant
Group: University of the West Indies, [email protected]

Proper citation: Angel Justiz-Vaillant 2020. Determination of IgG concentration by the Mancini test.. protocols.io https://dx.doi.org/10.17504/protocols.io.bj92kr8e Copy   

•   Source:

Authors: Diogo Provete

Proper citation: Diogo Provete 2016. Buccopharyngeal morphology of tadpoles in Scanning Electron Microscope. protocols.io https://dx.doi.org/10.17504/protocols.io.e4ebgte Copy   

•   Source:

Authors: Tyler Alioto
Group: SINGEK_ITN

Proper citation: Tyler Alioto 2018. Single Amplified Genome Assembly. protocols.io https://dx.doi.org/10.17504/protocols.io.nccdasw Copy   

•   Source:

Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: Allows for rapid generation of 3’ transcriptomic-NGS-ready- single-cell-libraries from pools of cells.Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Proper citation: Allen Institute for Brain Science 2021. 10Xv2 RNASeq Sample Processing. protocols.io https://dx.doi.org/10.17504/protocols.io.bq68mzhw Copy   

•   Source:

Authors: Gary Cline, John Stack
Group: Mouse Metabolic Phenotyping Centers
Summary: Procedure followed to detect the concentration of creatinine in serum, plasma, and urine.

Proper citation: Gary Cline, John Stack 2019. Yale - Blood and Urine Creatinine. protocols.io https://dx.doi.org/10.17504/protocols.io.y2dfya6 Copy   

•   Source:

Authors: Daniel Marchal

Proper citation: Daniel Marchal 2018. pH tolerance assay for Vibrio natriegens. protocols.io https://dx.doi.org/10.17504/protocols.io.prjdm4n Copy   

•   Source:

Authors: This protocol was modified by Sabrina Geraci-Yee and is from: Pozarowski P, Darzynkiewicz Z. 2004. Analysis of cell cycle by flow cytometry. Methods Mol Bio. 281:301-311.
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab
Summary: Sabrina Geraci-Yee modified this protocol from: Pozarowski P., Darzynkiewicz Z. 2004. Analysis of cell cycle by flow cytometry. Methods Mol. Bio. 281:301-311.

Proper citation: This protocol was modified by Sabrina Geraci-Yee and is from: Pozarowski P, Darzynkiewicz Z. 2004. Analysis of cell cycle by flow cytometry. Methods Mol Bio. 281:301-311. 2017. Staining Labyrinthulomycetes with Propidium Iodide. protocols.io https://dx.doi.org/10.17504/protocols.io.hfjb3kn Copy   

•   Source:

Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Serum levels of LDL-cholesterol reflect cholesterol metabolism and are associated with cardiovascular disease.

Proper citation: Jason Kim 2019. U Mass - Cholesterol (LDL). protocols.io https://dx.doi.org/10.17504/protocols.io.xt9fnr6 Copy   

•   Source:

Authors: G-Biosciences
Group: G-Biosciences
Summary: For routine protein assays, we recommend that researchers generate their own CB-X™ Tables or standard curves. This is a one time commitment to ensure user specific results and allows single tube assays to be performed each time as opposed to generating new calibration plots for each assay. We recommend using a protein standard similar to you protein of interest or a purified source of your protein.The supplied CB-X™ tables may result in some inconsistencies due to the type of cuvettes or microtiter plates used. CB-X™ Table for Spectrophotometer readings were measured with 1cm path length cuvettes against deionized water and CB-X™ Table for Microplate Reader readings were measured using Nunc™ Immuno 96 MicroWell™ Plates (MaxiSorp™ ) [Cat# 442404] against deionized water.

Proper citation: G-Biosciences 2016. Generating CB-X™ Tables and standard curves for CB-X Protein Assay Optimization. protocols.io https://dx.doi.org/10.17504/protocols.io.e4bbgsn Copy   

•   Source:

Authors: Nus Igem

Proper citation: Nus Igem 2019. Promega BacTiter-Glo Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.8ekhtcw Copy   

•   Source:

Authors: Isabelle Pagé, François Nougarou, Arnaud Lardon, Martin Descarreaux
Summary: The main goal of this project is to assess the reliability of thoracic spinal stiffness. To achieve this, 25 heartily participants (no significant thoracic pain in the past year) and 25 participants with chronic thoracic pain will participate in 2 experimental sessions separated by 24 to 48 hours. These sessions will occur at the Neuromecanic and Motor Control Laboratory of the Université du Québec à Trois-Rivières (Trois-Rivières, Québec, Canada). During these sessions, spinal stiffness will be measured at four thoracic spinal levels (T5 to T8) in a randomized sequence via a mechanical device using a servo-linear motor. The device indenter (metallic cylindrical cover with a rubber of 2 cm diameter) will gradually apply 45 N to the spinous process. The muscle activity and displacement data will be recorded during the procedures.

Proper citation: Isabelle Pagé, François Nougarou, Arnaud Lardon, Martin Descarreaux 2018. Changes in spinal stiffness with chronic thoracic pain: correlation with pain and muscle activity. protocols.io https://dx.doi.org/10.17504/protocols.io.twwepfe Copy   

•   Source:

Authors: Tomasz Krzywkowski, Mats Nilsson
Group: Molecular Diagnostics - Mats Nilsson Group, Neurodegeneration Method Development Community
Summary: This protocol is for in situ detection of mRNAs and single nucleotide polymorphisms using padlock probes and rolling circle amplification. In the accompanying publication, we take advantage of a single nucleotide variant within conserved ACTB mRNA to successfully differentiate human and mice co-cultured cells and apply following protocol to genotype PCDH X and Y homologs in human brain tissue sections. These are used as examples in the accompanying publication and custom padlock probes can be designed to allow for the targeting of own desired mRNA. We provide a method for automated characterization and quantitation of target mRNA in single cells or chosen tissue area. mRNA of interest, harboring a polymorphism, is first reverse-transcribed to cDNA. Allele specific padlock probes are hybridized to the cDNA target and enzymatically circularized maintaining a physical link with the parent mRNA molecule. Lastly, circularized probes are replicated in situ, using rolling circle amplification mechanism to facilitate detection.This protocol is taken directly from the accompanying publication. Further details and background information can be found in the cited published article.

Proper citation: Tomasz Krzywkowski, Mats Nilsson 2019. Detection of single nucleotide polymorphisms with padlock probes. protocols.io https://dx.doi.org/10.17504/protocols.io.wjhfcj6 Copy   

•   Source:

Authors: Alex Brown
Group: Reclone.org (The Reagent Collaboration Network)
Summary: About:This protocol has been optimized for recombinant expression of molony virus based reverse transcriptases (RT). The plasmid used contains a reverse transcriptase gene which is derived from Moloney Murine Leukemia Virus. The enzyme when expressed recombinantly can synthesizes a complementary DNA strand from single-stranded RNA, DNA, or an RNA:DNA hybrid.Enzyme Engineering:The enzyme contains point mutations to generate a highly processive, thermostable, and improved fidelity mutant reverse transcriptase. Details, incluiding the sequence of this enzyme are published and can be found in the references below and addgene link. The enzyme contains an active RNAse H domain and is highly thermostable. This MMLV construct contains the following mutations: D200N, L603W, T330P, L139P, E607K,D524G, E562Q, D583N and D653N. These appear to be the same set of mutations present in the Thermo Fisher Maxima H- RT, which is one of the best preforming RTs you can buy. The enzyme in our hands is extremely active. Moreover, while the MMLV RT is thermostable and will work up to 65°C it runs optimally at 42°C.The point muitations utilized here are currently filed under a provisional patent with Thermo Fisher Scientific. Commcercial use of this enzyme must go though Thermo Fisher Scientific.It is also possible to further engineer this enzyme with to increase thermostability and eliminate RNase H activity to generate an enzyme which closely mimicks Thermo Fisher Maxima H+ RT.Introducing mutations (D524G, E562Q, D583N and D653N) and/or (D524, D583, E562, H204, V223, T306, F309) are best suted eliminate RNase H activity completely, and it will increase the thermostability somewhat as wellComments:Reference 2 highlights several point mutations which can be introduced to eliminate Rnase H activity which may also enhance RT sensitivity. Thermo Product information brochure from 2013 suggest that this mutant or some combintation of other identified point mutations from reference 2 is Maxima RT, Maxima H-. The paper has also identified a few more useful point mutations which may further optimise MMLV-RTThis expression protocol can also implemented to express other MMLV based reverse transcriptases or diversely related retroviral RTs, provided the genes are in an expresison construct with an N- terminal 6-10 His Tag and are expressed under a T7 promoter.The plasmid used can be found on reclone.org and addgeneSkiBar H+ RT was synthesized as a gBlock from IDT and is codon optimized. Expressed in Rosetta DE3 his tag is on still but seems to work fine with it on.Tgatcc motif at MCS is a result of issues cloning in with BamHI. Had to use a compatible sticky end from BclI-HFProtein Properties: 79.9 kDaIsoelectric point (pI) : 7.77Charge at pH 7.0: 9.52Name:The name SkiBar H+ RT is an abbreviation of the first two first authors where the MMLV mutations used in the construct were originally discovered (Skirgaila & Baranauskas).References:Baranauskas, Aurimas Paliksa, Sigitas Alzbutas, Gediminas Vaitkevicius, Mindaugas Lubiene, Judita Letukiene, VirginijaBurinskas, Sigitas Sasnauskas, Giedrius Skirgaila, RemigijusGeneration and characterization of new highly thermostable and processive M-MuLV reverse transcriptase variantsProtein Engineering, Design and Selection (2012)Skirgaila, Remigijus Pudzaitis, Vaidas Paliksa, Sigitas Vaitkevicius, Mindaugas Janulaitis, ArvydasCompartmentalization of destabilized enzyme-mRNA-ribosome complexes generated by ribosome display: A novel tool for the directed evolution of enzymesProtein Engineering, Design and Selection (2013)

Proper citation: Alex Brown 2020. Recombinant Protein Expression of MMLV-RT H+ (SkiBar H+ RT) V2. protocols.io https://dx.doi.org/10.17504/protocols.io.bijzkcp6 Copy   

•   Source:

Authors: Sharona Sedighim, Olivier George
Group: George Lab

Proper citation: Sharona Sedighim, Olivier George 2020. Retro-orbital Bleeding for Rats. protocols.io https:// Copy   

•   Source:

Authors: Ethan Lippmann, Hannah Wilson, Emma Neal
Group: Neurodegeneration Method Development Community
Summary: Standardized single cell seeding protocol for Blood-Brain Barrier (BBB) differentiation.

Proper citation: Ethan Lippmann, Hannah Wilson, Emma Neal 2020. Single Cell Seeding of BBB Stem Cell Model. protocols.io https://dx.doi.org/10.17504/protocols.io.8j9hur6 Copy   

•   Source:

Authors: Doug Campbell, Natalie Donaher
Summary: Electrophoresis and transfer steps using the Life Technologies Bolt system as done by Campbell Lab for immunoquantitation of phytoplankton proteins.

Proper citation: Doug Campbell, Natalie Donaher 2017. Western Blotting using the Bolt system. protocols.io https://dx.doi.org/10.17504/protocols.io.jwqcpdw Copy   

•   Source:

Authors: Kailong Ma
Group: GigaScience Press, BGI
Summary: This protocol is used to clarity the process of RNA extraction for our Betta splendens genome.

Proper citation: Kailong Ma 2018. RNA extraction for the Betta splendens genome. protocols.io https://dx.doi.org/10.17504/protocols.io.qvfdw3n Copy   

•   Source: