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Authors: Fatma Ayhan, Genevieve Konopka
Group: Human Cell Atlas Method Development Community, Neurodegeneration Method Development Community
Summary: This protocol outlines our preparation of single-nuclei suspension from surgically acquired fresh human adult brain tissue.

Proper citation: Fatma Ayhan, Genevieve Konopka 2018. Nuclei Isolation from Human Brain Using Sucrose Gradient. protocols.io dx.doi.org/10.17504/protocols.io.scneave Copy   

•   Source: Protocols.io

Authors: Jaclyn Winter

Proper citation: Jaclyn Winter 2019. cpf1 cloning. protocols.io dx.doi.org/10.17504/protocols.io.8f4htqw Copy   

•   Source: Protocols.io

Authors: Blake Flood
Summary: Based on:https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0160509

Proper citation: Blake Flood 2019. Miracle Prep for Plasmid Isolation. protocols.io dx.doi.org/10.17504/protocols.io.8p2hvqe Copy   

•   Source: Protocols.io

Authors: Janina Brakel
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Collier Lab

Proper citation: Janina Brakel 2016. Labyrinthula growth medium. protocols.io dx.doi.org/10.17504/protocols.io.ehkbb4w Copy   

•   Source: Protocols.io

Authors: Saumel Perez Rodriguez, María De Jesús Ramírez-Lira, Tune Wulff, Bjørn Gunnar Voldbor, Octavio T Ramírez, Mauricio A Trujillo-Roldán, Norma A Valdez-Cruz
Summary: Subcellular fractionation of mammalian cells has been applied for the study of morphology, composition, structure and interactions between organelles, cellular and molecular biology and, more recently, the cell composition through omics approaches ⁠. Advantages of fractionation comprise but are not limited to obtaining fractions enriched in certain compartments for the study of cellular processes in vitro ⁠, locate and track proteins ⁠ and analyze post-translational modifications (PTM) of proteins processed along the secretory pathway ⁠ and protein composition of organelles⁠. Despite the existence of a wide variety of cell fractionation protocols and techniques, most of them are based on the identification or assignment of a certain isolated fraction to one or more subcellular compartments from the enrichment of this fraction in certain markers, the which are mostly protein. The quantification of these protein markers can be done through western blot, ELISA, enzymatic assays or proteomic studies. The present protocol is focused in the identification of proteins markers from several organelles, by WB, ELISA and enzymatic assays. The enrichment of isolated fractions in endoplasmic reticulum (ER), cytosol, nucleus, mitochondria, plasma membrane (PM), cis-Golgi and trans-Golgi can be assessed by detection of 78 kDa glucose-regulated protein (Grp78), glyceraldehyde 3-phosphate dehydrogenase (Gapdh), histone H3, heat shock protein 60 (Hsp60), flotilin 1, golgin A5 and golgin-97, respectively, by WB, and also by ELISA in case of golgin-97. Enrichment of peroxisomes in fractions collected from sucrose gradients can be assessed by a catalase assay adapted from . This protocol can be applied to any mammalian cell line during subcellular fractionation in order to quantify the enrichment of several organelles in the isolated fractions.

Proper citation: Saumel Perez Rodriguez, María De Jesús Ramírez-Lira, Tune Wulff, Bjørn Gunnar Voldbor, Octavio T Ramírez, Mauricio A Trujillo-Roldán, Norma A Valdez-Cruz 2020. Western blot, ELISA and enzymatic assays of reference proteins for subcellular fractionation . protocols.io dx.doi.org/10.17504/protocols.io.bgc4jsyw Copy   

•   Source: Protocols.io

Authors: Gregory L Boyer
Group: Metabolomics Protocols & Workflows
Summary: This standard operating procedure is used to determine and quantify microcystins and nodularin (intracellular and/or extracellular) in whole water samples or filter samples using HPLC-coupled with single quadrupole mass spectrometry (LC-MS). The method described here is a modification of the methods described in Boyer (2007) for particulate microcystins in water samples and does not use solid phase extraction. It was designed to use a 50% acidified methanolic extraction solvent, but is compatible with protocols for direct water analysis (US-EPA, 2016) and higher concentration methanolic extracts (Cerasino et al., 2017). It is not suitable for use with tissues samples or other samples with complex matrix interferences, such as soil or sediments. The method is designed for high throughput (1000-1500 injections per month) using a low-resolution (unit mass) mass spectrometer. It gives results of similar accuracy to the commonly used Abraxis ADDA ELISA with additional information on the microcystin congener profiles.

Proper citation: Gregory L Boyer 2020. LCMS-SOP Determination of Microcystins in Water Samples by High Performance Liquid Chromatography (HPLC) with Single Quadrupole Mass Spectrometry (MS). protocols.io dx.doi.org/10.17504/protocols.io.bck2iuye Copy   

•   Source: Protocols.io

Authors: Germán Alberto Téllez Ramírez, Lily Johanna Toro, Diana Carolina Henao, Juan David Rivera, Jhon Carlos Castaño Osorio
Group: Grupo de inmunología molecular
Summary: Esta prueba evalúa la actividad hemolítica de péptidos o moleculas sobre eritrocitos humanos como criterio de selectividad ante las células eucariotas. La realizacion de este protocolo fue posible gracias al apoyo del departamento administrativo de ciencia tecnología e innovacion, Colciencias a traves del proyecto 111356933173 convocatoria569-2012. 

Proper citation: Germán Alberto Téllez Ramírez, Lily Johanna Toro, Diana Carolina Henao, Juan David Rivera, Jhon Carlos Castaño Osorio 2017. PRUEBA DE HEMOLISIS DE ERITROCITOS HUMANOS. protocols.io dx.doi.org/10.17504/protocols.io.jh9cj96 Copy   

•   Source: Protocols.io

Authors: Marina Torres Betelli, Patrícia Sayuri Takazono, Caroline Ribeiro de Souza, Julia Ávila de Oliveira, Daniel Boari Coelho, Luis Augusto Teixeira
Summary: The scale for evaluation of compensatory arm and leg movements (CALM) was developed to evaluate balance stability recovery after unpredictable displacements of the support base in the mediolateral direction. The mediolateral perturbations are applied in three modes: rotation, translation or combined rotation-translation, to either side, in three platform peak velocities: 20º-20 cm/s (low), 30º-30cm/s (intermediate) or 0º-40 cm/s (high), keeping peak acceleration of 500o/s2(cm/s2) and displacement amplitude of 7o (cm) constant across perturbations. The factors perturbation mode (3), direction (2) and velocity (3) of platform displacement are combined to generate 18 distinct stance perturbations. To create the context of unpredictability, perturbations are pseudorandomly sequenced. To become aware of the stance perturbations, participants watch a video demonstrating a person responding to the different platform perturbations included in the protocol. After video watching, subjects are warmed up for 5 min. with global movements. Initial feet positions are marked on the support base with adhesive tapes (5-cm width). Other adhesive tape marks are fixed 15 cm away from the outer border of the feet to either side, in parallel to the feet orientation. The initial participant’s posture on the platform is keeping the Romberg’s stance, with the feet oriented forward, touching each other, maintaining both arms relaxed hanging beside the trunk, and palms of the hands lightly touching the upper legs. Participants wear a safety harness supported by two ropes tied at the shoulders height with the other end attached overhead. The participant’s aim across perturbations is to recover balance after support base displacements trying to maintain the initial body posture. Participants’ responses are filmed from behind using a commercial digital camera (Sony), for off-line analysis based on the CALM scale.

Proper citation: Marina Torres Betelli, Patrícia Sayuri Takazono, Caroline Ribeiro de Souza, Julia Ávila de Oliveira, Daniel Boari Coelho, Luis Augusto Teixeira 2019. Evaluation of stability of balance recovery from unpredictable perturbations through the compensatory arm and leg movements (CALM) scale. protocols.io dx.doi.org/10.17504/protocols.io.2p2gdqe Copy   

•   Source: Protocols.io

Authors: Yao Xiong, Qian Zhang, Jiaxiang Ye, Shan Pan, Lianying Ge
Summary: Conflicting results have been obtained regarding the association between X-ray repair cross complementation group 1 (XRCC1) and susceptibility to hepatocellular carcinoma (HCC). In this study, associations between HCC and three polymorphisms (Arg194Trp, Arg280His, and Arg399Gln) were evaluated using a meta-ananlysis approach. PubMed, Web of science, Cochrane Library, the Chinese National Knowledge Infrastructure, and the Wanfang standard database were systematically searched to identify all relevant case-control studies published through March 2018. A total of 32 case-control studies, including 13 that evaluated Arg194Trp, 14 that evaluated Arg280His, and 26 that evaluated Arg399Gln, were analyzed. In the entire study population, XRCC1 Arg399Gln was significantly associated not only with the overall risk of HCC (homozygous model, OR = 1.53, 95% CI: 1.34–1.76, P = 0.00; recessive model, OR = 1.31, 95% CI:1.15–1.49, P = 0.00), but also with the risk of HCC in Chinese patients (homozygous model, OR = 1.78, 95% CI: 1.53–2.08, P = 0.00; recessive model, OR = 1.47, 95% CI: 1.27–1.70, P = 0.00). Limiting the analysis to studies demonstrating Hardy–Weinberg equilibrium (HWE), the results were consistent and robust. Similarly, a significant association between XRCC1 Arg399Gln and HCC risk was found in healthy controls in the general population but not in hospital controls. Trial sequential analysis (TSA), the false-positive report probabilities (FPRP) and the combined genotype analysis revealed that XRCC1 Arg399Gln is mainly associated with susceptibility to liver cancer. However, there was no association between Arg194Trp or Arg280His and the risk of HCC. These results, indicating that the Arg399Gln polymorphism of XRCC1 is associated with the risk of HCC in the Chinese population, provide a basis for the development of improved detection and treatment approaches.

Proper citation: Yao Xiong, Qian Zhang, Jiaxiang Ye, Shan Pan, Lianying Ge 2018. Associations between three XRCC1 polymorphisms and the hepatocellular carcinoma risk: a meta-analysis of case-control studies. protocols.io dx.doi.org/10.17504/protocols.io.sqwedxe Copy   

•   Source: Protocols.io

Authors: Elena Casacuberta, Cristina Aresté, Omaya Dudin , Elena Casacuberta
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Multicellgenomelab, Emerging Organisms for Biology
Summary: Testing electroporation on Spaheroforma artica with two different electroporatorsNeon (invitrogen)Lonza (nucleofector)

Proper citation: Elena Casacuberta, Cristina Aresté, Omaya Dudin , Elena Casacuberta 2019. Unsucessful atemps on transfromation of the Icthyosporean Sphaeroforma artica. protocols.io dx.doi.org/10.17504/protocols.io.z6ef9be Copy   

•   Source: Protocols.io

Authors: Oliver Mead
Group: Solomon Lab - Australian National University
Summary: prepare 100 mL per 250 mL flask for each overnight culturefor     CZV8CS - liquid media for flask cultureprepare 100 mL per 250 mL flask for each overnight culture for 100 mL:                                300 mLCzapek-Dox liquid medium         4.54 g                                  13.62 g        centrifuged V8 juice                    20 mL                                   60 mLadd H20 to                                  95 mL                                   285 mL adjust to pH 6.0 transfer totransfer to flask(s) and autoclaveadd 5 mL sterile complete supplement to media after autoclavingComplete Supplement (CS)Bacto-casamino acids     20 g/lBacto-peptone                 20 g/lBacto-yeast extract         20g/lAdenine                           3 g/l 9Biotin                               0.02 g/lNicotinic acid                   0.02 g/lP-aminobenzoic acid       0.02 g/l 10Pyridoxine                        0.02 g/lThiamine                         0.02 g/l

Proper citation: Oliver Mead 2016. CZV8CS - liquid medium for fungal culture. protocols.io dx.doi.org/10.17504/protocols.io.f7cbriw Copy   

•   Source: Protocols.io

Authors: Martine Bangratz, Charlotte Tollenaere
Summary: Acid nucleic extraction of rice dried leaves by a CTAB method adapted from Li et al 2008. Li R, Mock R, Huang Q, Abad J, Hartung J, Kinard G. A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens. Journal of Virological Methods. 2008;154(1):48-55. doi: 10.1016/j.jviromet.2008.09.008.

Proper citation: Martine Bangratz, Charlotte Tollenaere 2020. Acid nucleic extraction from rice dried leaves . protocols.io dx.doi.org/10.17504/protocols.io.bcntiven Copy   

•   Source: Protocols.io

Authors: Ramawatar, Benjamin Schwessinger
Group: High molecular weight DNA extraction from all kingdoms
Summary: Most noncommercial tradition DNA extraction protocols result in a crude DNA preparation. If the DNA is intended to be used for a high-end application like Nanopore sequencing, it requires a thorough clean-up and size selection before it could be used for sequencing. Solid Phase Reversible Immobilisation (SPRI) magnetic beads is a quick and convenient way of purifying and size selecting intact double-stranded DNA from crude DNA. Most commercially available SPRI beads based DNA purification mix is quite expensive so our lab endeavored to develop an inexpensive beads mix which is as good as the commercially available ones. In this effort, our lab has optimized a beads mix for purifying and size selecting crude DNA extracted from eucalyptus and posted on protocol.io, https://www.protocols.io/edit/high-purity-high-molecular-weight-dna-extraction-f-n5ydg7w?step=16.However, this solution was not very effective in purifying crude DNA extracted from fungal material. DNA extracted from fungal material is highly viscous which is indicative of high levels of impurities in the DNA preparation. I tried to improve the beads mix for purifying rust DNA by adding 0.25 % (v/v) Tween-20 into the beads mix.I tested beads mix with Tween-20 to see if adding tween into the solution makes any difference in the recovery, purity and size selection.  Turns out that bead solutions with Tween-20 make big difference in the size selection and recovery of the DNA compared to the bead solutions without.  I calibrated/tested the bead solution with and without Tween-20 on the 1 kb DNA ladder to establish which DNA solution to beads volume ratio gives optimal recovery and size selection. We found that beads mix with 0.25 % Tween-20 works much better in size selection and recovery than beads mix without Tween-20. The best DNA to beads volume ratios were  1.0: 0.9 and 1.0: 1.0.

Proper citation: Ramawatar, Benjamin Schwessinger 2018. DNA size selection (>3-4kb) and purification of DNA using an improved homemade SPRI beads solution.. protocols.io dx.doi.org/10.17504/protocols.io.n7hdhj6 Copy   

•   Source: Protocols.io

Authors: Murat Eren and Bonnie Hurwitz
Group: Hurwitz Lab, MetaFunc Course
Summary: Adapted from http://merenlab.org/2016/06/22/anvio-tutorial-v2/

Proper citation: Murat Eren and Bonnie Hurwitz 2016. MG_HW9: Getting started with Anvi'o. protocols.io dx.doi.org/10.17504/protocols.io.gc3bsyn Copy   

•   Source: Protocols.io

Authors: Melissa Wilson Sayres
Summary: For more information and for the Spanish version of this protocol, please see here.The protocol is based on: http://www.scientificamerican.com/article/find-the-dna-in-a-banana-bring-science-home/.

Proper citation: Melissa Wilson Sayres 2016. Extracting DNA from bananas. protocols.io dx.doi.org/10.17504/protocols.io.esvbee6 Copy   

•   Source: Protocols.io

Authors: Dana Layo, Billy Y.B. Lau, Keerthi Krishnan
Summary: The purpose of this protocol is to outline the process by which we count high-intensity perineuronal nets (PNNs) in whole brain analysis. Specifically, high-intensity PNNs within the primary somatosensory cortex of adult mouse brain sections cut in a sagittal orientation. This protocol accompanies the paper “Lateralized expression of cortical perineuronal nets during maternal experience is dependent on MECP2” (https://doi.org/10.1101/787267).First, we align a digital map from the Franklin & Paxinos Mouse Brain Atlasonto individual brain sections. We then explain the process by which the S1 and its subregions areoutlined based on this atlas overlay. Finally, we outline the process of counting high-intensity PNNs within these regions.High-intensity PNNs are thought to be more mature structures due to a larger accumulation of the proteoglycans that make-up the structures1–3. We look at PNNs as a marker for changes in experience-dependent plasticity within the brain, where it can inhibit plasticity or solidify the changes that occurred in response to an experience.Video tutorial accompanying this protocol: Different regions of the mouse brain have differing densities of PNNs. Adult somatosensory cortex has the highest density based on what we have observed. This protocol can also be used to count all PNNs, just skip the contrast step (7.2) in PNN analysis.Citations1.Slaker, M. L., Harkness, J. H. & Sorg, B. A. A standardized and automated method of perineuronal net analysis using Wisteria floribunda agglutinin staining intensity.IBRO reports 1, 54–60 (2016).2.Carulli, D.et al.Composition of perineuronal nets in the adult rat cerebellum and the cellular origin of their components.The Journal of Comparative Neurology 494, 559–577 (2006).3.Foscarin, S.et al.Experience-Dependent Plasticity and Modulation of Growth Regulatory Molecules at Central Synapses.PLoS ONE 6, e16666 (2011).

Proper citation: Dana Layo, Billy Y.B. Lau, Keerthi Krishnan 2020. Mapping and Counting High-Intensity Perineuronal Nets in the Somatosensory Cortex. protocols.io dx.doi.org/10.17504/protocols.io.bcf8itrw Copy   

•   Source: Protocols.io

Authors: Franziska Aron, Gunnar Neumann, Guido Brandt
Group: WarinnerGroup, MPI-SHH Archaeogenetics
Summary: Protocol for the preparation of double-stranded genomic libraries for Illumina sequencing, optimised for ancient DNA (aDNA). This protocol generates adapter ligated DNA fragments that can be used in conjunction with downstream Indexing protocols.This protocol does not include UDG (USER) treatment, in order to retain molecular 'damage' in the form of deaminated cytosines characteristic of aDNA. This protocol is modified after Meyer & Kircher (2010) Cold Spring Harb. Protoc. (doi: 10.1101/pdb.prot5448).

Proper citation: Franziska Aron, Gunnar Neumann, Guido Brandt 2020. Non-UDG treated double-stranded ancient DNA library preparation for Illumina sequencing . protocols.io dx.doi.org/10.17504/protocols.io.bakricv6 Copy   

•   Source: Protocols.io

Authors: Linea Natalie Toksvang, Rikke Hebo Larsen, Thomas Leth Frandsen, Kjeld Schmiegelow, Cecilie Utke Rank
Summary: Hepatotoxicity was recognised as a complication of 6-thioguanine (6TG) in 1976. Since then, 6-TG associated hepatotoxicity in the form of acute sinusoidal obstruction syndrome (SOS), also known as veno-occlusive disease (VOD), has notably been reported in childhood acute lymphoblastic leukaemia (ALL), whereas chronic nodular regenerative hyperplasia (NRH) has been reported in adults with inflammatory bowel disease (IBD) as well as in childhood ALL. However, SOS and NRH should be considered part of a spectrum of microvascular disorders caused by endothelial injury. Nevertheless, the cellular mechanisms responsible for the sinusoidal damage, being pivotal to their development, remain to be established. 6TG-related SOS and NRH have been hypothesised to be dose-related, since they do not seem to occur with low cumulative doses. .justify:after { content: ""; display:inline-block; width: 100%; } The recent finding that higher levels of thioguanine nucleotides incorporated into leucocyte DNA (DNA-TGN) correlate to a lower relapse risk, calls for reappraisal of the feasibility of prolonged 6TG treatment for childhood ALL, while avoiding the risk of SOS and NRH. .justify:after { content: ""; display:inline-block; width: 100%; } .justify:after { content: ""; display:inline-block; width: 100%; } Objectives .justify:after { content: ""; display:inline-block; width: 100%; } - Primary objective: To assess the incidence of hepatotoxicity in patients treated with 6TG compared to 6MP or standard care. .justify:after { content: ""; display:inline-block; width: 100%; } - Secondary objective: To explore if a safe dose of 6TG can be established. .justify:after { content: ""; display:inline-block; width: 100%; }

Proper citation: Linea Natalie Toksvang, Rikke Hebo Larsen, Thomas Leth Frandsen, Kjeld Schmiegelow, Cecilie Utke Rank 2019. Hepatotoxicity during 6-thioguanine treatment: protocol for a systematic review. protocols.io dx.doi.org/10.17504/protocols.io.zjnf4me Copy   

•   Source: Protocols.io

Authors: Norio Yamamoto, Takahisa Ogawa, Masahiro Banno, Jun Watanabe, Tomoyuki Noda, Haggai Schermann, Toshifumi Ozaki

Proper citation: Norio Yamamoto, Takahisa Ogawa, Masahiro Banno, Jun Watanabe, Tomoyuki Noda, Haggai Schermann, Toshifumi Ozaki 2020. Protocol : Cement augmentation of internal fixation for trochanteric fracture: protocol for a systematic review and meta-analysis. protocols.io dx.doi.org/10.17504/protocols.io.bkg2ktye Copy   

•   Source: Protocols.io

Authors: Sean Seaver
Summary: How to make a 50 mg/ml Kanamycin Stock Solution

Proper citation: Sean Seaver 2014. How to make a 50 mg/ml Kanamycin Stock Solution. protocols.io dx.doi.org/10.17504/protocols.io.ci2ugd Copy   

•   Source: Protocols.io