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Authors: Adrian Clarke
Group: Protist Research to Optimize Tools in Genetics (PROT-G)

Proper citation: Adrian Clarke 2017. Skeletonema DNA extraction by Plant DNAzol. protocols.io https://dx.doi.org/10.17504/protocols.io.g6bbzan Copy   

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Authors: Ben Kuipers
Group: iGEM Wageningen 2019
Summary: This is a protocol used for the Dnase treatment after RNA isolation with the RNeasy mini kit. Protocol is based on the RNase-Free DNase Set and the RNeasy Mini Handbook 10/2013, Qiagen 'Appenix E: DNase Digestion of RNA before RNA Cleanup'.

Proper citation: Ben Kuipers 2019. DNase I treatment in solution (after RNA extraction). protocols.io https://dx.doi.org/10.17504/protocols.io.75ghq3w Copy   

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Authors: Sophie Adler, Kirstie Whitaker, Mira Semmelroch, Konrad Wagstyl
Summary: The MELD Project is an international collaboration aiming to create open-access, robust and generalisable tools for FCD detection. To this end, we will train a neural network classifier on MRI features from FCD patients from multiple centres worldwide.Protocol 1 details instructions for patient and control inclusion in the MELD Project as well as how to fill out the demographics csv file. 

Proper citation: Sophie Adler, Kirstie Whitaker, Mira Semmelroch, Konrad Wagstyl 2018. MELD Protocol 1 - Patient and Control Inclusion in the MELD Project. protocols.io https://dx.doi.org/10.17504/protocols.io.n3ddgi6 Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)

Proper citation: New England Biolabs 2020. Thermolabile Proteinase K Typical Reaction Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.7r6hm9e Copy   

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Authors: Mustapha Bittaye, Sarah Gilbert, Katie Ewer, Teresa Lambe
Group: Coronavirus Method Development Community
Summary: This Standard Operating Procedure (SOP) describes the techniques used to measure the titres of total human immunoglobulin G (IgG) responses against a full length recombinant clamped MERS-CoV spike glycoprotein (GP) in human plasma and serum samples. The measured antibody titres will serve to indicate the humoral immune response to a candidate ChAdOx1 MERS vaccine encoding the full length spike GP of the MERS-CoV in human subjects. This assay must be performed in accordance with the principles of the International Conference on Harmonisation Tripartite Guideline on Good Clinical Practice and the Human Tissue Act. Informed consent must be in place for samples to be tested in this assay.

Proper citation: Mustapha Bittaye, Sarah Gilbert, Katie Ewer, Teresa Lambe 2020. MERS-CoV Spike Glycoprotein (GP) – ELISA Jenner Clinical SOP Template. protocols.io https://dx.doi.org/10.17504/protocols.io.bgr6jv9e Copy   

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Authors: Christina Newman, Andrea Weiler, Dawn Dudley, Mitchell Ramuta, Amelia Haj, Shelby O'Connor, David O'Connor
Group: Coronavirus Method Development Community

Proper citation: Christina Newman, Andrea Weiler, Dawn Dudley, Mitchell Ramuta, Amelia Haj, Shelby O'Connor, David O'Connor 2020. RT-LAMP SARS-CoV-2 positive control stock. protocols.io https:// Copy   

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Authors: Mariam Awlia
Group: Salt Lab KAUST

Proper citation: Mariam Awlia 2019. Flame Photometry Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.6t6here Copy   

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Authors: Jana U'Ren, Lilly Moore
Group: U'Ren lab - Ecosystem Genomics
Summary: Some fungal isolates will embed into the solid media during growth, which makes it difficult to harvest agar-free mycelium for downstream applications such as DNA and RNA extraction. Here, we describe a protocol to (1) inoculate fungal isolates onto solid media with sterile cellophane overlays and (2) harvest the tissue for later DNA or RNA extraction.

Proper citation: Jana U'Ren, Lilly Moore 2021. Inoculating and harvesting fungal isolates on cellophane overlay . protocols.io https://dx.doi.org/10.17504/protocols.io.qtedwje Copy   

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Authors: Catherine Gohar, Ada de la Cruz
Group: Hillesland Lab

Proper citation: Catherine Gohar, Ada de la Cruz 2020. CCMA Coculture Media (Red Cap). protocols.io https://dx.doi.org/10.17504/protocols.io.bm8mk9u6 Copy   

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Authors: Jiri Hulcr, You Li, Sawyer Adams, Demian F Gomez
Group: Protocols Bark Beetle Mycobiome
Summary: This protocol describes the different media for fungal culturing.This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 https://doi.org/10.1007/s13199-020-00686-9.

Proper citation: Jiri Hulcr, You Li, Sawyer Adams, Demian F Gomez 2020. Media for Fungal Culturing. protocols.io https://dx.doi.org/10.17504/protocols.io.bnuwmexe Copy   

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Authors: Noreen Wauford

Proper citation: Noreen Wauford 2017. Cleavage Assay on Plate Reader. protocols.io https://dx.doi.org/10.17504/protocols.io.hd7b29n Copy   

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Authors: Alan Cone
Group: Ju Lab

Proper citation: Alan Cone 2016. PBS / DAPI 1:2000 Dilution Mixture. protocols.io https://dx.doi.org/10.17504/protocols.io.eifbcbn Copy   

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Authors: Zhujun Wei
Group: 2020 iGEM NEFU China
Summary: We use Tecan Spark® multimode microplate reader to measure absorbance.

Proper citation: Zhujun Wei 2020. Microplate reader operating procedure V.2. protocols.io https://dx.doi.org/10.17504/protocols.io.bnwymffw Copy   

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Authors: G-Biosciences
Group: G-Biosciences
Summary: The OmniPrep™ kit isolates high quality genomic DNA from many different species and tissue types including animal, plant, bacteria, yeast, fungi, whole blood, and cells in culture. DNA can be isolated from samples high in polysaccharides or other contaminants that are difficult to remove from the DNA preparations.This protocol is for use with cultured cells. Please refer to the appropriate protocol depending on your application.

Proper citation: G-Biosciences 2016. OmniPrep™ For High Quality Genomic DNA Extraction From Cultured Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.e5xbg7n Copy   

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Authors: Matema Imakumbili
Summary: This protocol describes how fertiliser solutions can be made and applied to already established plants in pot experiments. Before planting, fertiliser can be directly applied in solid form by evenly mixing it into the soil. This however cannot be done once the experiment has been initiated with plants already established. The fertiliser will hence have to be added to the soil with minimal disturbance and one way of doing this is by applying it in solution form. Adding fertiliser in solution form in pot experiments is particularly useful when second or third fertiliser applications have to be made for fertiliser applied in two or more split applications. Split applications of fertilisers are common for highly leachable fertilisers (e.g. urea), as this helps extend nutrient availability to growing plants. For various reasons, solid fertiliser application methods like banding or broadcasting are not suitable for pot experiments, hence the application of fertilisers to soils in established pot experiments in solution form. Fertiliser calculations are needed to determine the amount of fertiliser to add to pots. This protocol gives one example of a fertiliser calculation, but you will need to do your own reading for further understanding. See the attached document for guidance.

Proper citation: Matema Imakumbili 2019. Making and applying fertilisers in solution form in pot experiments. protocols.io https://dx.doi.org/10.17504/protocols.io.4ifgubn Copy   

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Authors: Fatma Gomaa, Zhuhong Li, Roberto Docampo, Peter Girguis, Virginia Edgcomb
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Bodo saltans kill curve protocol using G418 (Gentamicin)Gentamycin exhibits toxicity toward both eukaryotic and prokaryotic cells by disrupting ribosome function, thereby blocking the elongation step in protein synthesis.G418 is most commonly used as a selection agent for eukaryotic cells genetically engineered to express a neomycin resistance gene (NeoR), which is encoded by either transposon Tn601 (903) or Tn5.The resistant cells grow in medium containing G418, and may be used to establish stably transfected cell lines as all the non-resistant cells die due to G418 toxicity, typically within 6 – 14 days.To establish kill curve assay for saltans various concentrations of G418 were tested. The concentration range from 1 to 50 μg/mL for selection of eukaryotic, and then higher concentrations can be used for maintaining stable transfected cell line.Kill Curve Assay Harvest the B. saltans cells from a culture that is at peak density (1 – 3.0 x 105) by centrifugation at 800 x g for 5 min., discard the supernatant (medium). Replace the growth medium with fresh medium containing 0 – 50 μg/mL. For each concentration, test in triplicate. Using 6 wells plates, replace the medium in the wells every 3 – 4 days using fresh medium with the appropriate G418 concentration. Perform a daily visual inspection for evidence of toxicity, also cell count using hemocytometer. Note that the optimal dose of G418 for selection is the lowest one for which all cells have died after one week. A low dose is the concentration which has minimal effects on cells after 2 weeks of antibiotic selection. Conversely, a high dose is a concentration which is highly toxic to cells within 2-3 days of starting antibiotic selection. 5- The results indicate that a G418 concentration of  2 μg/mL kill the entire B. saltans population in 12 days which is considered enough time for selecting the resistant cells after transfection. G418 Sensitivity for B. saltans Cell Selection In all of our plasmids constructs we included the NeoR gene (Neomycine resistant gene) to select our transfected cells.Twenty-four hours after electroporation, the growth medium of transfected cells were supplemented with G418 antibiotic Solution (2 μg/mL) based on the kill curve results (above). 3. Replace the G418-containing medium every 3 – 5 days and examine cells for visual toxicity. Most non-transfected (non-resistant) cells will die within 10- 12 days, leaving the transfected cells to expand.Once cells grow to high confluence, they may be maintained, or frozen as a polyclonal line or plated by limited dilution to select for single clones.Replace the growth medium with fresh medium containing 0 – 50 μg/mL. For each concentration, test in triplicate. Using 6 wells plates, replace the medium in the wells every 3 – 4 days using fresh medium with the appropriate G418 concentration. Perform a daily visual inspection for evidence of toxicity, also cell count using hemocytometer. Note that the optimal dose of G418 for selection is the lowest one for which all cells have died after one week. A low dose is the concentration which has minimal effects on cells after 2 weeks of antibiotic selection. Conversely, a high dose is a concentration which is highly toxic to cells within 2-3 days of starting antibiotic selection. 5- The results indicate that a G418 concentration of  2 μg/mL kill the entire B. saltans population in 12 days which is considered enough time for selecting the resistant cells after transfection. G418 Sensitivity for B. saltans Cell Selection In all of our plasmids constructs we included the NeoR gene (Neomycine resistant gene) to select our transfected cells.Twenty-four hours after electroporation, the growth medium of transfected cells were supplemented with G418 antibiotic Solution (2 μg/mL) based on the kill curve results (above). 3. Replace the G418-containing medium every 3 – 5 days and examine cells for visual toxicity. Most non-transfected (non-resistant) cells will die within 10- 12 days, leaving the transfected cells to expand.Once cells grow to high confluence, they may be maintained, or frozen as a polyclonal line or plated by limited dilution to select for single clones.Using 6 wells plates, replace the medium in the wells every 3 – 4 days using fresh medium with the appropriate G418 concentration. Perform a daily visual inspection for evidence of toxicity, also cell count using hemocytometer. Note that the optimal dose of G418 for selection is the lowest one for which all cells have died after one week. A low dose is the concentration which has minimal effects on cells after 2 weeks of antibiotic selection. Conversely, a high dose is a concentration which is highly toxic to cells within 2-3 days of starting antibiotic selection. 5- The results indicate that a G418 concentration of  2 μg/mL kill the entire B. saltans population in 12 days which is considered enough time for selecting the resistant cells after transfection. G418 Sensitivity for B. saltans Cell Selection In all of our plasmids constructs we included the NeoR gene (Neomycine resistant gene) to select our transfected cells.Twenty-four hours after electroporation, the growth medium of transfected cells were supplemented with G418 antibiotic Solution (2 μg/mL) based on the kill curve results (above). 3. Replace the G418-containing medium every 3 – 5 days and examine cells for visual toxicity. Most non-transfected (non-resistant) cells will die within 10- 12 days, leaving the transfected cells to expand.Once cells grow to high confluence, they may be maintained, or frozen as a polyclonal line or plated by limited dilution to select for single clones.Note that the optimal dose of G418 for selection is the lowest one for which all cells have died after one week. A low dose is the concentration which has minimal effects on cells after 2 weeks of antibiotic selection. Conversely, a high dose is a concentration which is highly toxic to cells within 2-3 days of starting antibiotic selection. 5- The results indicate that a G418 concentration of  2 μg/mL kill the entire B. saltans population in 12 days which is considered enough time for selecting the resistant cells after transfection. G418 Sensitivity for B. saltans Cell Selection In all of our plasmids constructs we included the NeoR gene (Neomycine resistant gene) to select our transfected cells.Twenty-four hours after electroporation, the growth medium of transfected cells were supplemented with G418 antibiotic Solution (2 μg/mL) based on the kill curve results (above). 3. Replace the G418-containing medium every 3 – 5 days and examine cells for visual toxicity. Most non-transfected (non-resistant) cells will die within 10- 12 days, leaving the transfected cells to expand.Once cells grow to high confluence, they may be maintained, or frozen as a polyclonal line or plated by limited dilution to select for single clones.5- The results indicate that a G418 concentration of  2 μg/mL kill the entire B. saltans population in 12 days which is considered enough time for selecting the resistant cells after transfection.G418 Sensitivity for B. saltans AB1G418 (μg/ml) Cells survival (days) 250 2 320 2 415 3 510 4 65 6 73 8 82 12 91 >16 100 >16 A1G418 (μg/ml) A1Cell survival (days) Cell Selection In all of our plasmids constructs we included the NeoR gene (Neomycine resistant gene) to select our transfected cells.Twenty-four hours after electroporation, the growth medium of transfected cells were supplemented with G418 antibiotic Solution (2 μg/mL) based on the kill curve results (above). 3. Replace the G418-containing medium every 3 – 5 days and examine cells for visual toxicity. Most non-transfected (non-resistant) cells will die within 10- 12 days, leaving the transfected cells to expand.Once cells grow to high confluence, they may be maintained, or frozen as a polyclonal line or plated by limited dilution to select for single clones.3. Replace the G418-containing medium every 3 – 5 days and examine cells for visual toxicity. Most non-transfected (non-resistant) cells will die within 10- 12 days, leaving the transfected cells to expand.Once cells grow to high confluence, they may be maintained, or frozen as a polyclonal line or plated by limited dilution to select for single clones.

Proper citation: Fatma Gomaa, Zhuhong Li, Roberto Docampo, Peter Girguis, Virginia Edgcomb 2018. G418 Kill curve protocol . protocols.io https://dx.doi.org/10.17504/protocols.io.sh4eb8w Copy   

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Authors: Wakana Shoda, Naohiro Nomura, Fumiaki Ando, Hideaki Tagashira, Takahiro Iwamoto, Akihito Ohta, Kiyosih Isobe, Takayasu Mori, Koichiro Susa, Eisei Sohara, Tatemitsu Rai, Shinichi Uchida
Summary: This protocol was used for the experiment conducted in " Sodium-calcium exchanger 1 is the key molecule for urinary potassium excretion against acute hyperkalemia"

Proper citation: Wakana Shoda, Naohiro Nomura, Fumiaki Ando, Hideaki Tagashira, Takahiro Iwamoto, Akihito Ohta, Kiyosih Isobe, Takayasu Mori, Koichiro Susa, Eisei Sohara, Tatemitsu Rai, Shinichi Uchida 2019. intracellular calcium assay . protocols.io https://dx.doi.org/10.17504/protocols.io.baihicb6 Copy   

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Authors: Joe Horecka and Angela M. Chu
Group: Genomes to Natural Products Network
Summary: A protocol for yeast colony PCR, starting with intact cells. Can be used to analyze genomic and plasmid DNA, with PCR products up to 2 kb being no problem. It doesn't get easier than this: pick a small amount of cells from a plate into water, lyse cells at 99C for 5 min. Use the lysed cells as template by adding to 1/10th final volume in a PCR. We use this protocol for diagnostics (Phire HS II enzyme) and for Sanger sequencing (Phusion HS II or NEB Q5 enzyme). The main reasons why others might not get this protocol to work could be due to using too many cells and/or not using pure water for the cell lysis.  Good luck, and have fun! See the 'BEFORE START' section of this protocol for photographs that show how to pick cells from a colony and make a suspension.  See the 'WARNINGS' section for important points and trouble shooting. 

Proper citation: Joe Horecka and Angela M. Chu 2017. Yeast Colony PCR: It doesn't get any easier than this!. protocols.io https://dx.doi.org/10.17504/protocols.io.gzwbx7e Copy   

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Authors: Omneya Ahmed Osman, Alexander Eiler, Mats Töpel, Tomas Larsson, Johan Andersson
Group: eDNAsolutions
Summary: This protocol explain the qPCR assay for detecting the pathogenic fungus Batrachochytrium dendrobatidis in environmental DNA samples and metabarcoding of amphibians species.

Proper citation: Omneya Ahmed Osman, Alexander Eiler, Mats Töpel, Tomas Larsson, Johan Andersson 2019. Detection of Batrachochytrium dendrobatidis (Bd) and amphibians DNA metabarcoding. protocols.io https://dx.doi.org/10.17504/protocols.io.94gh8tw Copy   

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Authors: Julie Huber, Caroline Fortunato
Group: Center for Dark Energy Biosphere Investigations, Huber Lab
Summary: This is a protocol for carrying out RNA Stable Isotope Probing experiments using seawater or vent fluids to examine microbial bicarbonate uptake. It was developed at Axial Seamount to examine autotrophy in deep-sea hydrothermal vent fluids. We have used modified versions in seawater, cold crustal environments, and other hydrothermal vents as well.

Proper citation: Julie Huber, Caroline Fortunato 2017. RNA Stable Isotope Probing Experimental Set Up. protocols.io https://dx.doi.org/10.17504/protocols.io.iuzcex6 Copy   

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