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Authors: Sarah Hu
Group: Caron Lab - Protistan Ecology
Summary: This protocol provides step-by-step instructions for implementing a perl script to easily automate quality checking of raw tag sequences. This was specifically constructed for the numerous 18S diversity projects (which target the V4 hypervariable specific for evaluting protistan diversity).It is highly recommended that you follow along this Github repo: https://github.com/shu251/QC_steps_V4_tagsequencing
Proper citation: Sarah Hu 2017. Microbial eukaryotic 18S tag-sequence processing/QC - V4 region (automating). protocols.io dx.doi.org/10.17504/protocols.io.j9bcr2n Copy
Authors: Alexandra Ehl, David Frommholz, Nadine Stefanczyk
Summary: Purification Guide for the Isolation of Antibodies with ChroPlate Filtration Plates by DALEX Biotech.Easy and quick high throughput antibody purification from various sources and species.Each well of the ChroPlate has a binding capacity of > 1 mg (tested with human polyclonal Ig, binding varies between species and clones).The proprietary resin does not shrink or swell in aqueous buffers.High pressure stability.pH stability short term 2 - 8, long term 3 - 8.Excellent thermal stability up to 15 minutes at 80 °C in aqueous buffers at neutral pH.Can be dried for long term storage (80 °C for > 2 h).
Proper citation: Alexandra Ehl, David Frommholz, Nadine Stefanczyk 2019. ChroPlate - ProteinG. protocols.io dx.doi.org/10.17504/protocols.io.8qthvwn Copy
Authors: Marco Clementi, Mario Schietroma, Emanuele Lezoche, Francesco Carlei, Alessio Agnifili, Federico Sista, Sara Colozzi, Paola Mariani, Francesco Masedu, Stefano Guadagni.
Summary: Background: VEGF is elevated in plasma of colon cancer patients and increases after surgery. This study's purpose was to determine plasma VEGF levels before, during, and after open and laparoscopic colorectal surgery performed for left colon cancer (CD).Patients and methods: a multicenter, prospective, observational, non-randomized study was planned to evaluate samples obtained preoperatively, intraoperatively, and on postoperative days (1, 3, 7, 15, 30). VEGF-A165 values were determined via ELISA. Statistical analysis was a repeated Anova with interactions.Results: the recruitment finished in 4 years (71 patients). Baseline plasma VEGF-A165 mean values (pVmv) resulted not significantly different in patients submitted to laparoscopy (125.4 pg/ml ± 47.7) and laparotomy (116.3 pg/ml ± 28.9). Intraoperatively, the highest difference was registered at the end of operations. In the 30-days postoperative period, the pVmv resulted significantly lower (G-G=0.45) in patients submitted to laparoscopy than in patients submitted to laparotomy (F= 144.6, pConclusions: the trial supports the hypothesis that cancer and surgical trauma are related to production of VEGF-A165 and demonstrates that open surgery, in comparison to laparoscopy, is a stronger promoter of VEGF release.
Proper citation: Marco Clementi, Mario Schietroma, Emanuele Lezoche, Francesco Carlei, Alessio Agnifili, Federico Sista, Sara Colozzi, Paola Mariani, Francesco Masedu, Stefano Guadagni. 2017. VASCULAR ENDOTHELIAL GROWTH FACTOR PLASMA LEVELS IN PATIENTS WITH CANCER OF THE LEFT COLON SUBMITTED TO OPEN OR LAPAROSCOPIC SURGERY. protocols.io dx.doi.org/10.17504/protocols.io.mdzc276 Copy
Authors: Fernando Espinola
Summary: This protocol describes the steps used to analyze shotgun sequences deposited in in Integrated Microbial Genomes and Microbiomes (IMG/M ) by using functional anotation evidences. KEGG orthology terms and pathways are used as evidence.The abundance of each of the corresponding biomarker genes is estimated by calculating the proportion of amino acid sequences assigned to the KO of interest (estimated gene copies, assembled and unassembled metagenomes, as retrieved from IMG/M) and normalized with respect to the total number of sequences assigned to KOs in each metagenome.
Proper citation: Fernando Espinola 2018. Bioinformatic analysis of biomarker genes using metagenomic shotgun sequence datasets. protocols.io dx.doi.org/10.17504/protocols.io.u32eyqe Copy
Authors: Prakash Lab
Group: Foldscope
Summary: The printed instructions accompanying each Foldscope kit are very detailed and easy to follow. Even easier is this excellent 14-minute video tutorial for the assembly. I didn't know about the video when I was assembling the Foldscope, and despite carefully following the printed instructions, I still managed to make a few embarrassing mistakes. I am sharing the protocol below with the comments on these mistakes, so others can avoid them. Hopefully, more new users will comment, pinpointing the assembly steps that turned out to be error-prone for them.
Proper citation: Prakash Lab 2016. Foldscope™ Assembly Instructions. protocols.io dx.doi.org/10.17504/protocols.io.fjwbkpe Copy
Authors: Igem Dusseldorf
Summary: This protocol enables direct, one-pot fatty acid extraction and derivatisation of plant and bacterial samples for preparation for GC-MS analysis.The protocol was established by the Weber lab for plant seedlings and seeds, but was successfully used for cyanobacteria and E.coli as well.
Proper citation: Igem Dusseldorf 2019. Fatty acid extraction and derivatisation. protocols.io dx.doi.org/10.17504/protocols.io.79jhr4n Copy
Authors: Imen Lassadi
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Attempts to transfect Hematodinium was performed using a plasmid that will express eGFP under the control of DVNP regulatory sequences. See attachment for plasmid information.
Proper citation: Imen Lassadi 2019. Plasmid used for transfection trails of Hematodinium . protocols.io dx.doi.org/10.17504/protocols.io.4nigvce Copy
Authors: Eva Feldman
Group: Diabetic Complications Consortium
Summary: Summary:Phenotyping of Rodents for the Presence of Diabetic Neuropathy In man, the development of diabetic neuropathy is dependent on both the degree of glycemic control and the duration of diabetes. Diabetic neuropathy is a progressive disorder, with signs and symptoms that parallel the loss of nerve fibers over time. Consequently, assessments of neuropathy in mice are not performed at one time point, but are characterized at multiple time points during a 6 month period of diabetes. The degree of diabetes is evaluated in 2 ways: tail blood glucose measured following a 6 hour fast and glycated hemoglobin levels. The initial degree of neuropathy is screened using the methods discussed below. Detailed measures of neuropathy are employed when the initial screening instruments indicate a profound or unique phenotypic difference. This document contains protocols used by the DiaComp staff to examine and measure diabetic neuropathy at the whole animal, tissue and cellular levels.Diabetic Complication:Reference: AFIP Laboratory Methods in Histotechnology, 1994, p. 16, 53-55.
Proper citation: Eva Feldman 2019. Neuropathy Phentoyping Protocols - Hematoxylin and Eosin Staining. protocols.io dx.doi.org/10.17504/protocols.io.3jsgkne Copy
Authors: Haseena Khan
Proper citation: Haseena Khan 2017. Protoplast Isolation from Zymoseptoria tritici. protocols.io dx.doi.org/10.17504/protocols.io.k2mcyc6 Copy
Authors: Angel Justiz-Vaillant, Norma McFarlane-Anderson
Group: University of the West Indies, [email protected]
Summary: This SpAG ELISA can be used to detect specific antibodies in various animal species including human, goat, donkey, mouse, rat, dog, cow, horse, rabbit, chicken, monkey, pig and hamster [1].1. Kelly PJ, Tagwira M, Matthewman L, Mason PR, Wright EP. Reactions of sera from laboratory, domestic and wild animals in Africa with protein A and a recombinant chimeric protein AG.Comp Immunol Microbiol Infect Dis. 1993;16(4):299-305. doi:10.1016/0147-9571(93)90159-3
Proper citation: Angel Justiz-Vaillant, Norma McFarlane-Anderson 2020. Enzyme linked immunosorbent assay for investigating the binding of Protein-AG (SpAG) to immunoglobulins.. protocols.io dx.doi.org/10.17504/protocols.io.bjppkmmn Copy
Authors: Bonnie Hurwitz, Ken Youens-Clark
Group: VERVE Net, Hurwitz Lab
Summary: Long-standing questions in marine viral ecology are centered on understanding how viral assemblages change along gradients in space and time. However, investigating these fundamental ecological questions has been challenging due to incomplete representation of naturally occurring viral diversity in single gene- or morphology-based studies and an inability to identify up to 90% of reads in viral metagenomes (viromes). In this protocol, I describe how to use an annotation- and assembly-free strategy for comparative metagenomics that combines shared k-mer and social network analyses (regression modeling). This robust statistical framework enables visualization of complex sample networks and determination of ecological factors driving community structure. This tutorial describes a protocol to reproduce work from the Pacific Ocean virome comprised of 32 viromes from diverse sites in the Pacific Ocean. "Modeling ecological drivers in marine viral communities using comparative metagenomics and network analyses" (July 7, 2014, doi: 10.1073/pnas.1319778111, PNAS July 22, 2014 vol. 111 no. 29 10714-10719)Code is freely available at Github.
Proper citation: Bonnie Hurwitz, Ken Youens-Clark 2016. Modeling ecological drivers in marine viral communities using comparative metagenomics and network analyses. protocols.io dx.doi.org/10.17504/protocols.io.efgbbjw Copy
Authors: Dennis Dienst, Oliver Mantovani, Pia Lindberg
Group: CyanoWorld, Metabolomics Protocols & Workflows, Chemistry Method Development Community
Summary: This is a quick guide for the preparation of (-)-α-bisabolol samples and external standards in dodecane for GC analysis. This protocol has been established in the Lindberg lab at Ångström laboratory (Uppsala University) for direct analysis of dodecane-based ex-situ extracts from cyanobacterial strains producing (-)-α-bisabolol. The protocol includes the preparation of stock solutions for the internal standard β-caryophyllene (BCP) and the external standard. The preparation of a fresh external standard series is suggested for each sample analysis. The calibration curve is required for quantification of (-)-α-bisabolol in the samples of interest. This protocol is used in combination with the following GC protocol:dx.doi.org/10.17504/protocols.io.kj2cuqe
Proper citation: Dennis Dienst, Oliver Mantovani, Pia Lindberg 2019. (-)-α-Bisabolol GC sample preparation. protocols.io dx.doi.org/10.17504/protocols.io.xiufkew Copy
Authors: Ariel Rabines, Rob Lampe, Andrew Allen
Group: A.E. Allen Lab
Summary: A mostly automated protocol for extraction of total RNA from seawater filtered onto a Sterivex filter (Cat. No. SVGP0150). Reagents come from the Macherey-Nagel NucleoMag RNA kit (Cat. No. 744350). Automated liquid handling is performed on an eppendorf EpMotion 5075t with multi-channel pipettes. This protocol assumes that the Sterivex is sealed with tube sealant on the male end and a male-luer lock plug on the female end. Using this protocol, we routinely extract ~80 samples at a time.The protocol has been modified to increase to an 800 µL starting volume. This normally results in less Lysis Buffer MR1, Binding Buffer MR2, and Reducing Agent TCEP supplied than is needed. Extra reagents can and should be purchased separately. The kit manual can be found here: https://www.mn-net.com/media/pdf/23/c2/23/Instruction-NucleoMag-RNA.pdfAdditional notes from the manufacturer on using the kit with the epMotion can be found here: https://www.mn-net.com/media/pdf/bd/7e/53/AN-NucleoMag-RNA-epMotion5075.pdf . Note that we have several additional modifications to their protocol.The epMotion program is attached here.
Proper citation: Ariel Rabines, Rob Lampe, Andrew Allen 2020. Sterivex RNA extraction. protocols.io dx.doi.org/10.17504/protocols.io.bd9ti96n Copy
Authors: Matthew Sullivan Lab
Group: VERVE Net, Sullivan Lab
Summary: Preparation of the trace metal mixture for addition to seawater for the cultivation of marine cyanobacteria, Prochlorococcus and Synechococcus
Proper citation: Matthew Sullivan Lab 2015. Cyanobacteria Trace Metal Mixture (CTMM). protocols.io dx.doi.org/10.17504/protocols.io.c8nzvd Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This is the protocol for Protein Expression Using BL21(DE3) Competent E. coli (C2527). For large scale, please follow this protocol variant.
Proper citation: New England Biolabs 2014. Protein Expression Using BL21(DE3) (C2527). protocols.io dx.doi.org/10.17504/protocols.io.cjqumv Copy
Authors: Frank Aylward
Summary: This is an example of a simple read mapping workflow. It is designed to be performed via the command line on an Ubuntu 16.06 OS. After completing this tutorial you should:1) Have a practical understanding of how read mapping analyses are performed in the command line.2) Understand the basics of how to process SAM and BAM files.3) Be able to calculate depth of coverage of a contig/scaffold/chromosome in a query read dataset.
Proper citation: Frank Aylward 2018. Introduction to read mapping. protocols.io dx.doi.org/10.17504/protocols.io.piadkae Copy
Authors: Mitchell Finger, Michael Lyon, Judy Northill, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services
Summary: This real-time TaqMan-MGB RT-PCR protocol aimed to amplify measles virus (MeV) strains and not other viruses.Michael Lyon and Mitchell Finger designed the assay in 2009 using Primer Express software.The method was later published by Greg Smith in 2010 (see below).The assay targets the fusion (F) gene region and is designed as a qualitative test for investigating MeV infection of humans.This was a past assay that we no longer in use. For our favoured Measles virus TaqMan test, please refer to the MeV N TaqMan protocol.
Proper citation: Mitchell Finger, Michael Lyon, Judy Northill, Ian Mackay 2019. Measles virus TaqMan RT-PCR (F gene; no longer in regular use; see Guidelines). protocols.io dx.doi.org/10.17504/protocols.io.8b9hsr6 Copy
Authors: Steven Wilhelm, Samantha Coy
Group: Samantha R Coy's Protocols
Summary: Ultracentrifugation in the WX Ultra Series can support only 30 mL samples at a time, and therefore do not significantly concentrate viruses. Tangential flow filtration enables viral concentration of large volumes that can then be purified using sucrose density gradients. Contact Dr. Steven Wilhelm ([email protected]) or Samantha Coy ([email protected]) for additional information regarding this protocol.
Proper citation: Steven Wilhelm, Samantha Coy 2020. Concentrating Viruses by Tangential Flow Filtration. protocols.io dx.doi.org/10.17504/protocols.io.bbagiibw Copy
Authors: Lukas Snoek, Tinka Beemsterboer
Group: Spinoza Centre, REC-L
Summary: This protocol lists all the steps necessary to run your MRI experiment/data acquisition safely and in a way that yields high-quality data. Moreover, if you use the centre's QC/preprocessing service, it lists the steps necessary to make sure we can convert the data into BIDS and run them through the QC/preprocessing pipelines.
Proper citation: Lukas Snoek, Tinka Beemsterboer 2018. During data acquisition. protocols.io dx.doi.org/10.17504/protocols.io.p5vdq66 Copy
Authors: Chisholm Lab
Group: VERVE Net, Sullivan Lab
Proper citation: Chisholm Lab 2016. NATURAL SEAWATER-BASED PRO99 MEDIUM. protocols.io dx.doi.org/10.17504/protocols.io.c7zzp5 Copy
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