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A-Tailing with Taq Mixture Resource Report Resource Website |
New England Biolabs | 10.17504/protocols.io.crxv7m | New England Biolabs (NEB) | New England Biolabs | https://www.neb.com/protocols/2013/11/01/a-tailing-with-taq-polymerase | 1 | 2015 | New England Biolabs 2015. A-Tailing with Taq Mixture. protocols.io dx.doi.org/10.17504/protocols.io.crxv7m | 2021-03-29 03:08:32 | |||
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Isolation of mouse islet cells, culture with heparan sulfate mimetics and flow cytometry analysis of beta cell viability Resource Report Resource Website |
Sarah Popp, Sarita Dhounchak, Charmaine Simeonovic | 10.17504/protocols.io.bmgjk3un | Isolated mouse islets were dispersed into single cells using Accutase (Millipore; 250 µl/500 islets). 4-8 x 104 islet cells were transferred to individual wells of a 96 well culture plate (CELLSTAR, Greiner Bio-one) for immediate staining for flow cytometry analysis or for culture prior to staining. Isolated mouse islet cells were cultured in the presence or absence of the HS mimetics heparin (a highly sulfated HS analogue from porcine intestinal mucosa) or PI-88 (Progen Pharmaceuticals Limited,) at 50 mg/ml for 2 days in 5% CO2, 95% air at 37ºC. In some studies islet cells were acutely treated with 30% H2O2 (Chem-Supply) as a source of reactive oxygen species (ROS) for 5 min on day 0 or after culture for 2 days with/without HS mimetics. Damaged and dying islet cells were assessed using Calcein-AM (Calcein; 0.04 µM; Invitrogen)/Propidium iodide (PI; 2.5 µg/ml; BD Biosciences) or by Sytox green (31.25 nmol/L; Invitrogen, Molecular Probes) uptake. BD LSR Fortessa flow cytometer BD FACS DIVA software (version 8) were used to collect events and Flow Jo software (version 10.0.7, TreeStar Inc.) was used to analyse the intensity of fluorescence staining. | The Australian National University, The Australian National University, The Australian National University | 1 | 2020 | Sarah Popp, Sarita Dhounchak, Charmaine Simeonovic 2020. Isolation of mouse islet cells, culture with heparan sulfate mimetics and flow cytometry analysis of beta cell viability. protocols.io dx.doi.org/10.17504/protocols.io.bmgjk3un | 2021-03-29 03:08:32 | ||||
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Case - Lipid Analysis Assay by GC-mass spectrometry Resource Report Resource Website |
Henri Brunengraber | 10.17504/protocols.io.yehftb6 | Mouse Metabolic Phenotyping Centers | SummaryA known quantity of tissue / plasma is hydrolyzed and extracted after adding known amounts of internal standards: eg. heptadecanoic acid and cholesterol-d7. Fatty acids / cholesterol are analyzed as their trimethylsilyl derivatives using gas chromatography-electron impact ionization mass spectrometry (GCMS) (note: this protocol outlines the processing for palmitate and cholesterol; other fatty acids and sterols can be assayed using this preparation, see refs 1,2).References: 1. Triglyceride synthesis in epididymal adipose tissue: contribution of glucose and non-glucose carbon sources. Bederman IR, Foy S, Chandramouli V, Alexander JC, Previs SF. J Biol Chem. 2009, 284(10):6101-8. 2. Influence of diet on the modeling of adipose tissue triglycerides during growth. Brunengraber DZ, McCabe BJ, Kasumov T, Alexander JC, Chandramouli V, Previs SF. Am J Physiol Endocrinol Metab. 2003, 285(4):E917-25. | Case Western Reserve University | https://mmpc.org/shared/document.aspx?id=274&docType=Protocol | 1 | 2019 | Henri Brunengraber 2019. Case - Lipid Analysis Assay by GC-mass spectrometry. protocols.io dx.doi.org/10.17504/protocols.io.yehftb6 | 2021-03-29 03:08:32 | ||
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U Mass - Hemoglobin A1c Resource Report Resource Website |
Jason Kim | 10.17504/protocols.io.xy3fpyn | Mouse Metabolic Phenotyping Centers | Summary:This experiment provides the quantification of multiple cytokines and chemokines using multiplexed-Luminex technology based on beads containing specific antibodies. Serum cytokine levels reflect chronic or acute inflammation, and circulating cytokines and chemokines are altered in obesity. Cytokines Panel I include IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, KC (IL-8 homologue), IL-9, IL-10 (interleukin-10), IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, TNFα (tumor necrosis factor alpha), IFNγ (interferon gamma), IP-10 (interferon gamma-induced protein 10; CXCL-10), Eotaxin (CCL-11), G-CSF (granulocyte colony stimulating factor), GM-CSF (granulocyte macrophage colony stimulating factor), LIF (leukemia inhibitory factor), LIX (LPSinduced CXC chemokine), MCP-1 (monocyte chemotactic protein-1; CCL-2), M-CSF (macrophage colony stimulating factor), MIG (monokine induced by gamma interferon; CXCL9), MIP-1α (macrophage inflammatory protein alpha; CCL-3), MIP-1β (macrophage inflammatory protein beta; CCL-4), and MIP-2/RANTES (macrophage inflammatory protein 2alpha; CXCL-2). A service can be requested for all or any combination of listed cytokines/chemokines for customized multiplexed Luminex assay. | University of Massachusetts | https://mmpc.org/shared/document.aspx?id=160&docType=Protocol | 1 | 2019 | Jason Kim 2019. U Mass - Hemoglobin A1c. protocols.io dx.doi.org/10.17504/protocols.io.xy3fpyn | 2021-03-29 03:08:33 | ||
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Online microscopy and histology resources Resource Report Resource Website |
Zbigniew Mikulski | 10.17504/protocols.io.ux8exrw | La Jolla Institute Microscopy Core | There is a wealth of microscopy-related information online. This document lists the ones we know and like. Click on the document tab to see the list! | La Jolla Institute for Immunology | 3 | 2018 | Zbigniew Mikulski 2018. Online microscopy and histology resources. protocols.io dx.doi.org/10.17504/protocols.io.ux8exrw | 2021-03-29 03:08:32 | |||
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Chimeric Protein-LAG and Peptostreptococcal protein L sandwich ELISA Resource Report Resource Website |
Angel Justiz-Vaillant | 10.17504/protocols.io.btbaniie | Carbon | This ELISA is used to study the interactions between protein-LAG (PLAG) and Peptostreptococcal protein-L (SpL) with different immunoglobulin preparations from mammalian and avian species. | University of the West Indies St. Augustine | 1 | 2021 | Angel Justiz-Vaillant 2021. Chimeric Protein-LAG and Peptostreptococcal protein L sandwich ELISA. protocols.io dx.doi.org/10.17504/protocols.io.btbaniie | 2021-03-29 03:08:31 | |||
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Newport Beach Pier Weekly Sampling Protocol Resource Report Resource Website |
Jayme Smith, Emily Eggleston | 10.17504/protocols.io.pcudiww | Caron Lab - Protistan Ecology | University of Southern California, University of Southern California | 1 | 2018 | Jayme Smith, Emily Eggleston 2018. Newport Beach Pier Weekly Sampling Protocol. protocols.io dx.doi.org/10.17504/protocols.io.pcudiww | 2021-03-29 03:08:31 | ||||
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A+ Media for Marine Phytoplankton Resource Report Resource Website |
Dr. Steven Wilhelm | 10.17504/protocols.io.ibncame | Protist Research to Optimize Tools in Genetics (PROT-G), The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs) | Please contact Dr. Steven Wilhelm ([email protected]) for additional information regarding this protocol.Modified from Wilhelm SW and CG Trick. 1995. Physiological profiles of Synechococcus (Cyanophyceae) in iron-limiting continuous cultures. Journal of Phycology, 31:79-85. | 1 | 2017 | Dr. Steven Wilhelm 2017. A+ Media for Marine Phytoplankton. protocols.io dx.doi.org/10.17504/protocols.io.ibncame | 2021-03-29 03:08:32 | ||||
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Coil Resistance Testing Apparatus for VUSE ALTO Resource Report Resource Website |
Qutaiba Saleh, Edward Hensel, Risa Robinson | 10.17504/protocols.io.bibnkame | Respiratory Technologies Lab | Measuring coil resistance of Electronic Nicotine Delivery Systems (ENDS) accurately is critical in any research studying the characteristics of electronic cigarettes and their effects on the performance of these devices. It has been shown in several papers that changing coil resistance has the potential to change the Hazardous and Potentially Hazardous Constituents (HPHC) of emissions and consequently health effects on users. This protocol describes how to build a test apparatus for coil resistance measurement for ENDS. This apparatus mimics the geometrical and electrical characteristics of the ENDS and thus provides accurate measurements of the effective coil resistance. The steps shown in this protocol are illustrated for creating a VUSE ALTO test apparatus, but the general idea can be applied to other devices. |
MDPI and ACS Style Saleh, Q.M.; Hensel, E.C.; Robinson, R.J. Method for Quantifying Variation in the Resistance of Electronic Cigarette Coils. Int. J. Environ. Res. Public Health 2020, 17, 7779. |
Respiratory Technologies Lab, Rochester Institute of Technology, Respiratory Technologies Lab, Rochester Institute of Technology, Respiratory Technologies Lab, Rochester Institute of Technology | https://www.mdpi.com/1660-4601/17/21/7779/htm | 1 | 2020 | Qutaiba Saleh, Edward Hensel, Risa Robinson 2020. Coil Resistance Testing Apparatus for VUSE ALTO. protocols.io dx.doi.org/10.17504/protocols.io.bibnkame | 2021-03-29 03:08:32 | |
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High-Density Penetrating Microelectrode Recordings from Anesthetized Feline Dorsal Root Ganglia Resource Report Resource Website |
Zachariah Sperry, Kyounghwan Na, Mihaly Vöröslakos, Saman Parizi, James Jun, Tim M. Bruns, Euisik Yoon, John P. Seymour | 10.17504/protocols.io.w5nfg5e | SPARC | Dorsal root ganglia (DRG) are components of spinal roots containing sensory cell bodies, and hold significant promise as neural recording sites for sensory neuroprostheses.This protocol details the fabrication and use of a high-density flexible polyimide electrode array into the sacral DRG of an anesthetized feline for neural recordings. | University of Michigan - Ann Arbor, University of Michigan - Ann Arbor, University of Szeged, University of Michigan - Ann Arbor, Flatiron Institute, University of Michigan - Ann Arbor, University of Michigan - Ann Arbor, University of Michigan - Ann Arbor | https://doi.org/10.1101/435800 | 1 | 2021 | Zachariah Sperry, Kyounghwan Na, Mihaly Vöröslakos, Saman Parizi, James Jun, Tim M. Bruns, Euisik Yoon, John P. Seymour 2021. High-Density Penetrating Microelectrode Recordings from Anesthetized Feline Dorsal Root Ganglia. protocols.io dx.doi.org/10.17504/protocols.io.w5nfg5e | 2021-03-29 03:08:32 | ||
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Protocols for "Chromosome-level genome assembly of the humpback puffer, Tetraodon palembangensis" Resource Report Resource Website |
Rui Zhang, Chang Li, Mengjun Yu, Xiaoyun Huang, Mengqi Zhang, Shanshan Liu, Shanshan Pan, Weizhen Xue, Congyan Wang, Chunyan Mao, He Zhang, Guangyi Fan | 10.17504/protocols.io.bkczksx6 | GigaScience Press | The humpback puffer,Tetraodon palembangensis, also known as Pao palembangensis, is a species of poisonous freshwater pufferfish mainly distributed in Southeast Asia (Thailand, Laos, Malaysia and Indonesia). Despite interesting biological features, such as its very inactive nature, tetrodotoxin production and body expansion mechanisms, molecular research on the humpback puffer is still rare because of the lack of a high-quality reference genome. Here, we reported a first chromosome-level genome assembly of an adult humpback puffer, of which the genome size is 362 Mb with ~1.78 Mb contig N50 and ~15.8 Mb scaffold N50s. Based on the genome, ~61.5Mb (18.11%) repeat sequences were also identified, and totally 19,925 genes were annotated, 99.20% of which could be predicted with function using protein-coding function databases. Finally, a phylogenetic tree was constructed with single-copy gene families from ten teleost fishes. The humpback puffer genome will be a valuable genomic resource to illustrate possible mechanisms of tetrodotoxin synthesis and tolerance, providing clues for future detailed studies of biological toxins. | BGI-Qingdao, BGI-Shenzhen, Qingdao 26655¬¬5, China., BGI-Qingdao, BGI-Shenzhen, Qingdao 26655¬¬5, China., BGI-Qingdao, BGI-Shenzhen, Qingdao 26655¬¬5, China., BGI-Qingdao, BGI-Shenzhen, Qingdao 26655¬¬5, China., BGI-Qingdao, BGI-Shenzhen, Qingdao 26655¬¬5, China., BGI-Qingdao, BGI-Shenzhen, Qingdao 26655¬¬5, China., BGI-Qingdao, BGI-Shenzhen, Qingdao 26655¬¬5, China., BGI-Qingdao, BGI-Shenzhen, Qingdao 26655¬¬5, China., BGI-Qingdao, BGI-Shenzhen, Qingdao 26655¬¬5, China., BGI-Qingdao, BGI-Shenzhen, Qingdao 26655¬¬5, China., BGI-Qingdao, BGI-Shenzhen, Qingdao 26655¬¬5, China., BGI-Qingdao, BGI-Shenzhen, Qingdao 26655¬¬5, China. | 1 | 2020 | Rui Zhang, Chang Li, Mengjun Yu, Xiaoyun Huang, Mengqi Zhang, Shanshan Liu, Shanshan Pan, Weizhen Xue, Congyan Wang, Chunyan Mao, He Zhang, Guangyi Fan 2020. Protocols for "Chromosome-level genome assembly of the humpback puffer, Tetraodon palembangensis". protocols.io dx.doi.org/10.17504/protocols.io.bkczksx6 | 2021-03-29 03:08:32 | |||
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HTAPP_Depletion of CD45+ cells from ovarian cancer ascites single cell suspensions for single-cell RNA-Seq Resource Report Resource Website |
Benjamin Izar, Parin Shah, Mei-Ju Su, Isaac Wakiro, Sara Napolitano, Jingyi Wu, Sébastien Vigneau, Asaf Rotem, Orit Rozenblatt-Rosen, Bruce Johnson, Aviv Regev | 10.17504/protocols.io.bhbij2ke | NCIHTAN | Many ovarian cancer ascites samples have a high proportion of CD45-expressing cells (>90%) (CD45+) and a low proportion (For the Human Tumor Atlas Pilot Project (HTAPP) this protocol was used to deplete CD45+ immune cells from single-cell suspensions obtained from ovarian cancer ascites, and is included as a step in the "HTAPP_Processing human ovarian cancer ascites to a single-cell suspension for single-cell RNA-seq" protocol, also available in protocols.io. It is adapted from the CD45 MicroBeads MACS Separation protocol from Miltenyi Biotec (Cat No 130-045-801). Description of this protocol and guidance for testing and selecting methods for processing different tumor and sample types can be found in Slyper et al. | Dana-Farber Cancer Institute;Human Tumor Atlas Pilot Project, Dana-Farber Cancer Institute;Human Tumor Atlas Pilot Project, Dana-Farber Cancer Institute;Human Tumor Atlas Pilot Project, Dana-Farber Cancer Institute, University of California, Santa Barbara, Dana-Farber Cancer Institute;Human Tumor Atlas Pilot Project, Dana-Farber Cancer Institute;Human Tumor Atlas Pilot Project, Dana-Farber Cancer Institute;Human Tumor Atlas Pilot Project, Broad Institute;Human Tumor Atlas Pilot Project, Dana-Farber Cancer Institute;Human Tumor Atlas Pilot Project, Broad Institute;Human Tumor Atlas Project;Massachusetts Institute of Technology;Howard Hughes Medical Institute | 2 | 2020 | Benjamin Izar, Parin Shah, Mei-Ju Su, Isaac Wakiro, Sara Napolitano, Jingyi Wu, Sébastien Vigneau, Asaf Rotem, Orit Rozenblatt-Rosen, Bruce Johnson, Aviv Regev 2020. HTAPP_Depletion of CD45+ cells from ovarian cancer ascites single cell suspensions for single-cell RNA-Seq. protocols.io dx.doi.org/10.17504/protocols.io.bhbij2ke | 2021-03-29 03:08:31 | |||
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ddRADSeq in a Field Setting Resource Report Resource Website |
Mrinalini Watsa, Gideon Erkenswick, Aaron Pomerantz, Stefan Prost | 10.17504/protocols.io.y2efybe | This protocol begins with blood stored in Longmire's solution and has as its goal, the sequencing of genomic DNA from several individuals onto a single MinION sequencing run. It is based on a protocol outlined by Thrasher et al. (2018) for the same process on a large number of warbler samples, but it differs from this original protocol in a few key ways:Since we ultimately were targeting sequencing by a MinION sequencer, the ultimate processing steps reflect library preparation for runs on this device, instead of Illumina sequencers.For the same reason, we also size selected larger fragments than can be sequenced on a MiSeq platform.This protocol is also meant to only analysed a few individuals (20 or less) since the MinION read coverage is lower than that of a MiSeq.The entire protocol was carried out in a field laboratory in the southeastern Peruvian Amazon, and therefore many of the luxuries a institutional laboratory can afford were not present to usWe used gel-based size selection in the absence of methods such as Blue Pippin.We stored blood on FTA cards and in Longmire's solution in our sampling program. We discovered that yields from FTA cards, regardless of including large numbers of hole punches per extraction, simply were too low for the sequencing goals of this project. Therefore, we include the protocol of a WGA amplification step for these low samples. Longmire's solution produced higher yields, but some times, multiple extractions might need to be pooled together with an SPRI bead cleanup to be fully effective. | University of Missouri - Saint Louis; Washington University in Saint Louis; Field Projects International, University of Missouri - Saint Louis; Field Projects International, University of California, Berkeley, CA, USA, Department of Integrative Biology; Marine Biological Laboratory, Woods Hole, MA - USA, LOEWE-Center for Translational Biodiversity Genomics, Senckenberg Museum, 60325 Frankfurt, Germany; South African National Biodiversity Institute, National Zoological Garden, Pretoria 0184, South Africa | 3 | 2019 | Mrinalini Watsa, Gideon Erkenswick, Aaron Pomerantz, Stefan Prost 2019. ddRADSeq in a Field Setting. protocols.io dx.doi.org/10.17504/protocols.io.y2efybe | 2021-03-29 03:08:32 | ||||
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Haematoxylin-Eosin stain for cryosections Resource Report Resource Website |
Simon Blanchoud | 10.17504/protocols.io.bru8m6zw | Blanchoud lab, UNIFR | University of Fribourg | 1 | 2021 | Simon Blanchoud 2021. Haematoxylin-Eosin stain for cryosections. protocols.io dx.doi.org/10.17504/protocols.io.bru8m6zw | 2021-03-29 03:08:32 | ||||
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Rab29 fast exchange mutants: characterization of a challenging Rab GTPase Resource Report Resource Website |
Rachel C. Gomez, Edmundo G. Vides, Suzanne Pfeffer | 10.17504/protocols.io.bffrjjm6 | Rab29 has been implicated in multiple membrane trafficking processes with no described effectors or regulating proteins. Its fast nucleotide exchange rate and inability to bind GDI in cytosol make it a unique and poorly understood Rab. Because the conventional, “GTP-locked” Rab mutation does not have the desired effect in Rab29, we present here the use of a fluorescence-based assay to characterize novel mutants that display faster nucleotide exchange rates, allowing for GEF-independent Rab29 activation. | Stanford University School of Medicine, Stanford University School of Medicine, Stanford University School of Medicine | 1 | 2020 | Rachel C. Gomez, Edmundo G. Vides, Suzanne Pfeffer 2020. Rab29 fast exchange mutants: characterization of a challenging Rab GTPase. protocols.io dx.doi.org/10.17504/protocols.io.bffrjjm6 | 2021-03-29 03:08:32 | ||||
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Electroporation transformation of FITC-dextran into Oxyrrhis marina - an early branching dinoflagellate. Resource Report Resource Website |
Nicholas Irwin, Elisabeth Hehenberger, Patrick Keeling | 10.17504/protocols.io.ginbude | Protist Research to Optimize Tools in Genetics (PROT-G) | Here we describe a protocol for transforming Oxyrrhis marina with FITC-labelled dextran (a DNA analog) by electroporation. This protocol is reproducible, and results in a transformation efficiency of around 50%.We have confirmed the viability of transformed cells by allowing them to recover in the dark (and thus maintain fluorescence intensity) and then by observing fluorescent cells swimming normally, days after transformation (see the video here). As of yet we have been unable to demonstrate the actual uptake of plasmid DNA. We have tried using rhodamine labelled plasmids to demonstrate this but the results were inconclusive due to issues with cellular autofluorescence. We have also tried this method with a variety of expression plasmids encoding different promoters. For example, we tried the MOE promoter from Perkinsus marinus (a closely related species) and the generic eukaryotic CMV promoter. However no GFP fluorescence was detected in these samples. We have also tried transforming in vitro synthesized GFP encoding mRNA using this protocol. As of yet, no GFP production has been detected, but this may reflect non-cannonical translational systems in dinoflagellates as the same mRNA could be translated in starfish embryos.Although the development of expression systems in dinoflagellates remains a challenge, our results suggest that O. marina may be amenable to genetic transformation. | University of British Columbia, University of British Columbia, University of British Columbia | 1 | 2016 | Nicholas Irwin, Elisabeth Hehenberger, Patrick Keeling 2016. Electroporation transformation of FITC-dextran into Oxyrrhis marina - an early branching dinoflagellate.. protocols.io dx.doi.org/10.17504/protocols.io.ginbude | 2021-03-29 03:08:32 | |||
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UCP-1 (Abcam ab10983) immunohistochemical protocol Resource Report Resource Website |
Bianca Hemmeryckx, Dries Bauters, H. Roger Lijnen | 10.17504/protocols.io.j9scr6e | Increasing energy expenditure by stimulating thermogenesis through activation of brown adipose tissue (BAT) and/or induction of browning of white adipose tissue (WAT) is considered a promising strategy to treat/prevent obesity and related metabolic diseases. Whereas WAT is adapted to store energy as triglycerides, BAT produces heat (non-shivering thermogenesis). In brown adipocytes, the uncoupling protein-1 (UCP-1) regulates conversion of energy into heat by uncoupling ATP production from mitochondrial respiration. Also in WAT adaptive UCP-1 positive adipocytes (brown in white: brite or beige) can arise, predominantly in subcutaneous (s) WAT. This browning of WAT is enhanced by exposure to cold temperatures. | Bauters D, Bedossa P, Lijnen HR, Hemmeryckx B (2018) Functional role of ADAMTS5 in adiposity and metabolic health. PLoS ONE 13(1): e0190595. doi: 10.1371/journal.pone.0190595 | KU Leuven, KU Leuven, KU Leuven | https://doi.org/10.1371/journal.pone.0190595 | 1 | 2018 | Bianca Hemmeryckx, Dries Bauters, H. Roger Lijnen 2018. UCP-1 (Abcam ab10983) immunohistochemical protocol. protocols.io dx.doi.org/10.17504/protocols.io.j9scr6e | 2021-03-29 03:08:32 | ||
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U Mass - Lipid metabolism Resource Report Resource Website |
Jason Kim | 10.17504/protocols.io.x3gfqjw | Mouse Metabolic Phenotyping Centers | Lipid metabolism is estimated by measuring systemic clearance of [1-14C] palmitate following a bolus injection in awake mice. Lipid metabolism is altered in obese mice. | University of Massachusetts | https://mmpc.org/shared/document.aspx?id=145&docType=Protocol | 1 | 2019 | Jason Kim 2019. U Mass - Lipid metabolism. protocols.io dx.doi.org/10.17504/protocols.io.x3gfqjw | 2021-03-29 03:08:32 | ||
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Efficacy of metformin in patients with breast cancer receiving chemotherapy or endocrine therapy：Systematic review and meta-analysis (protocol) Resource Report Resource Website |
Kayoko Morio, Yasuko Kurata, Nobuko Kawaguchi-Sakita, Akihiro Shiroshita, Yuki Kataoka | 10.17504/protocols.io.bmzak72e | Department of Pharmacy, Kobe University Hospital 7-5-2, Kusunoki-cho, Chuo-ku, Kobe, 650-0017, Japan, Department of Pharmacy, Okayama University Hospital, 2-5-1 Shikata-cho, Kita-ku, Okayama 700-8558, Japan, Department of Clinical Oncology, Kyoto University Hospital, 54 Shogoin-Kawaracho, Sakyo-ku, Kyoto 606-8507, Japan, Department of respiratory medicine, Ichinomiya-nishi hospital, Japan, Hospital Care Research Unit, Hyogo Prefectural Amagasaki General Medical Center, Higashinaniwa-cho 2-17-77, Amagasaki 660-8550, Japan | 2 | 2020 | Kayoko Morio, Yasuko Kurata, Nobuko Kawaguchi-Sakita, Akihiro Shiroshita, Yuki Kataoka 2020. Efficacy of metformin in patients with breast cancer receiving chemotherapy or endocrine therapy：Systematic review and meta-analysis (protocol). protocols.io dx.doi.org/10.17504/protocols.io.bmzak72e | 2021-03-29 03:08:30 | |||||
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Multi-step high purity high molecular weight DNA extraction protocol from challenging fungal tissues Resource Report Resource Website |
Ramawatar Nagar, Benjamin Schwessinger | 10.17504/protocols.io.rzkd74w | High molecular weight DNA extraction from all kingdoms | This protocol is based on the fact that we struggled for a long time to obtain high purity DNA from the fungal material, especially from several rust species. We finally thought we made it when we obtained DNA with perfect QC measures using a CTAB based DNA precipitation. Yet we still got low yields caused by high amount of 'active feedback' during the sequencing run. We and many others had similar observations that awesome looking DNA doesn't sequence well (https://www.protocols.io/groups/awesome-DNA-from-all-kingdoms-of-life/discussions/awesome-dna-purity-measures-but-quickly-dying-pores). In this protocol, we combined several ideas we accumulated over the last 1.5 years since we started working on HMW DNA extractions for Nanopore sequencing. -> Different precipitants have different affinities for different contaminants.-> Some contaminants may have a higher affinity and lower solubility in NaCl/PEG/SPRI 'clean' up steps.-> Adding enzyme cocktails during the extraction may help to get rid of some contaminants. We combined all these three steps in the current protocol as combinatorial testing is currently cost prohibitive. It may well be that one of these steps is already enough.These ideas are laid out in more detail below and in publication soon to come. Our general recommendation is to test different buffer conditions and precipitants and if necessary combine them in a sequential manner. We hypothesize that different precipitants, e.g. NaCl/PEG, isopropanol, ethanol, or CTAB, display varying affinities for precipitating different contaminants. By applying them in a sequential manner it may be possible to obtain clean DNA via preferential precipitation of DNA over contaminants. In addition, in this newly developed protocol, we add enzyme mixes to the extraction buffer containing pectinases and cellulases reducing the amount of co-purifying contaminants from the fungal tissue. In case of other tissue types, different enzymes may have to be tested. It is important to add these enzymes during the extraction and not apply them to the final DNA suspension as most are not completely pure enzyme preparations and contain traces of DNAase activity that degrades the DNA when applied in simple solutions like TE buffer. We (see above) and many others have reported that NaCl/PEG-SPRI bead solutions are not always ideally suited to clean up DNA as contaminants simply co-precipitate. Following a similar logic of preferential precipitation, We hypothesize that is possible to first precipitate contaminants onto SPRI beads at low NaCl/PEG concentrations when HMW DNA stays in solution. In a subsequent step, DNA can be precipitated out of the remaining supernatant by increasing NaCl/PEG concentration adding more of the initial NaCl/PEG-SPRI beads solution. Contaminants with higher affinity to SPRI beads and lower solubility than DNA can thereby be removed from the solution.It is important to mention that we have had DNA preparations that fulfilled all our recommended quality control criteria but did not sequence well on the MinION. This was likely caused by ‘invisible’ contaminants. However, applying a combination of the above-suggested approaches enabled us to overcome this problem with our latest protocol. | Australian National University, Australian National University | 1 | 2018 | Ramawatar Nagar, Benjamin Schwessinger 2018. Multi-step high purity high molecular weight DNA extraction protocol from challenging fungal tissues. protocols.io dx.doi.org/10.17504/protocols.io.rzkd74w | 2021-03-29 03:08:31 |
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