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Authors: jessechao
Group: University of British Columbia

Proper citation: jessechao 2020. Variant functionalization by localization. protocols.io https://dx.doi.org/10.17504/protocols.io.bn68mhhw Copy   

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Authors: Ben Tully
Group: ECOGEO
Summary: Quality impact results is the universal first step for all sequencing methods. This protocol is for sequencing results in FASTQ format. FastQC allows for visualization of quality scores. Sequence trimming allows for removal of residual primer sequences and increases overall quality scores. Trimmomatic does both. CutAdapt cuts adapters from sequences. Sickle creates a sliding window for quality and length trimming of FASTQ files.Open this protocol inside the virtual machine (details in 'Start Instructions') for easy copy, paste of commands into the command line terminal window.

Proper citation: Ben Tully 2016. ECOGEO 'Omics Training: 2.0 Quality Control. protocols.io https://dx.doi.org/10.17504/protocols.io.fixbkfn Copy   

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Authors: Angel Justiz-Vaillant
Group: University of the West Indies, [email protected]
Summary: A protein that combines the binding capacity of SpA, SpG and SpL is not comercially available. It was created in my laboratory by combining these 3 immunoglobulin-binding proteins to horseradish peroxidase by the periodate method [1]. However, a mixture of SpA, SpG and SpL which are comercially available can have the same effect as universal reagent in immunodetection. 1. Vaillant AJ, McFarlane-Andersonv N, Wisdom B, Mohammed W, Vuma S, et al. (2013) Immunoglobulin-binding Bacterial Proteins (IBP) Conjugates and their Reactivity with Immunoglobulin in Enzyme-Linked Immunosorbent Assays (ELISA). J Anal Bioanal Tech 4: 175. doi:10.4172/2155-9872.1000175

Proper citation: Angel Justiz-Vaillant 2020. Universal Immunoblot analysis for investigating Protein-LAG (SpLAG)-binding to mammalian and avian immunoglobulins.. protocols.io https://dx.doi.org/10.17504/protocols.io.bjsdkna6 Copy   

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Authors: Treseder K, Holden S, Maltz M
Summary: DNA ExtractionExtract DNA from sample using the phenol/chloroform procedure or your kit of choice. We typically use the Mo Bio Power Soil DNA extraction kit for extracting DNA from soil and plant litter samples.Use the Nanodrop in the Martiny lab to estimate the concentration and purity of your DNA extraction.Dilute DNA to 10 ng/ul prior to PCR. PCR AmplificationPre-amplification checklist:All pipetting should be done with aerosol barrier, PCR certified pipet tips in a PCR work stationOn ice thaw primers, DNA and PCR master mixDecontaminate pipettors and interior surfaces of a PCR workstation with DNA/RNA Away or 10% bleach solution.UV irradiate PCR work station interior, pipettors and consumables for minimum 15 minAll samples should be amplified in triplicate.Include negative (no template) and positive (DNA isolated from mushroom) controls. Primer information:Forward primer:454 primer B + 'AG' linker + SSU817fGCCTTGCCAGCCCGCTCAGAGTTAGCATGGAATAATRRAATAGGA Reverse Primer:454 primer A + 12-base bar code + 'AC' linker + ssu1196rGCCTCCCTCGCGCCATCAG-12 base bar code-ACTCTGGACCTGGTGAGTTTCCExample barcode: ACACACTATGGCExample primer sequence: GCCTCCCTCGCGCCATCAGACACACTATGGCACTCTGGACCTGGTGAGTTTCC PCR Cocktail:22.5 ul PCR master mix (we use the Invitrogen Platinum PCR SuperMix)1 ul BSA0.75 ul 10 uM forward primer (all samples get the same forward primer)0.75 ul 10 uM reverse primer (all samples get a unique barcoded reverse primer)1 ul template DNA PCR cycle:Samples are initially denatured at 94°C for 10 min, then amplified using 30 cycles of 94°C for 45 sec, 52°C for 30 sec, and 72°C for 90 sec. A final extension of 10 min at 72°C is added at the end of the program to ensure complete amplification of the target region. Check for PCR product on a gel Amplicon CleaningCombine the triplicate PCR reactions into a single volumeClean PCR reactions with a PCR clean up kit. The MoBio UltraClean PCR clean up kit and the Invitrogen Purelink PCR purification kit have both worked well. Amplicon QuantificationUse the qubit system for quantifying DNA, with a few modifications.Instead of using the qubit assay tubes, prepare the samples in black microplates.Perform assay with 1 ul of PCR productRead the plates on the microplate reader in the Allison lab set at 485ex /530em. Estimate amplicon concentration using the standard curve. Amplicon PoolingCombine equal amounts of DNA per sample into single vessel (usually a 15 mL tube).Concentrate pooled amplicons using the Invitorgen Purelink PCR purification kitRun a gel to visualize pooled samplesDepending on how your pooled amplicons look on the gel, you may need to gel purify the pooled amplicons. Pyrosequencing is biased towards shorter reads. If you have a messy band on your gel, it is important to purify your samples to improve the quality of the DNA you will receive. We used the Qiagen QIAquick Gel Extraction Kit. We ran samples out on a 1.4 % gel. The main drawback to performing a gel extraction is that you will almost certainly lose some of your sample. You may need to pool additional PCR product and perform a second gel extraction to obtain enough sample for sequencing. Remove contaminants from gel purified samples using an Invitrogen Purelink PCR purification kit. Estimate final sample concentration and purityUse the qubit system for estimating sample concentration. The sequencing facility requires 2-3 ug of DNA for sequencing.Use the Nanodrop in the Martiny lab for estimating sample purity. Samples with an A260/A280 ratio of ~1.8 typically provide optimal sequencing results. Send sample for sequencingExtract DNA from sample using the phenol/chloroform procedure or your kit of choice. We typically use the Mo Bio Power Soil DNA extraction kit for extracting DNA from soil and plant litter samples.Use the Nanodrop in the Martiny lab to estimate the concentration and purity of your DNA extraction.Dilute DNA to 10 ng/ul prior to PCR. PCR AmplificationPre-amplification checklist:All pipetting should be done with aerosol barrier, PCR certified pipet tips in a PCR work stationOn ice thaw primers, DNA and PCR master mixDecontaminate pipettors and interior surfaces of a PCR workstation with DNA/RNA Away or 10% bleach solution.UV irradiate PCR work station interior, pipettors and consumables for minimum 15 minAll samples should be amplified in triplicate.Include negative (no template) and positive (DNA isolated from mushroom) controls. Primer information:Forward primer:454 primer B + 'AG' linker + SSU817fGCCTTGCCAGCCCGCTCAGAGTTAGCATGGAATAATRRAATAGGA Reverse Primer:454 primer A + 12-base bar code + 'AC' linker + ssu1196rGCCTCCCTCGCGCCATCAG-12 base bar code-ACTCTGGACCTGGTGAGTTTCCExample barcode: ACACACTATGGCExample primer sequence: GCCTCCCTCGCGCCATCAGACACACTATGGCACTCTGGACCTGGTGAGTTTCC PCR Cocktail:22.5 ul PCR master mix (we use the Invitrogen Platinum PCR SuperMix)1 ul BSA0.75 ul 10 uM forward primer (all samples get the same forward primer)0.75 ul 10 uM reverse primer (all samples get a unique barcoded reverse primer)1 ul template DNA PCR cycle:Samples are initially denatured at 94°C for 10 min, then amplified using 30 cycles of 94°C for 45 sec, 52°C for 30 sec, and 72°C for 90 sec. A final extension of 10 min at 72°C is added at the end of the program to ensure complete amplification of the target region. Check for PCR product on a gel Amplicon CleaningCombine the triplicate PCR reactions into a single volumeClean PCR reactions with a PCR clean up kit. The MoBio UltraClean PCR clean up kit and the Invitrogen Purelink PCR purification kit have both worked well. Amplicon QuantificationUse the qubit system for quantifying DNA, with a few modifications.Instead of using the qubit assay tubes, prepare the samples in black microplates.Perform assay with 1 ul of PCR productRead the plates on the microplate reader in the Allison lab set at 485ex /530em. Estimate amplicon concentration using the standard curve. Amplicon PoolingCombine equal amounts of DNA per sample into single vessel (usually a 15 mL tube).Concentrate pooled amplicons using the Invitorgen Purelink PCR purification kitRun a gel to visualize pooled samplesDepending on how your pooled amplicons look on the gel, you may need to gel purify the pooled amplicons. Pyrosequencing is biased towards shorter reads. If you have a messy band on your gel, it is important to purify your samples to improve the quality of the DNA you will receive. We used the Qiagen QIAquick Gel Extraction Kit. We ran samples out on a 1.4 % gel. The main drawback to performing a gel extraction is that you will almost certainly lose some of your sample. You may need to pool additional PCR product and perform a second gel extraction to obtain enough sample for sequencing. Remove contaminants from gel purified samples using an Invitrogen Purelink PCR purification kit. Estimate final sample concentration and purityUse the qubit system for estimating sample concentration. The sequencing facility requires 2-3 ug of DNA for sequencing.Use the Nanodrop in the Martiny lab for estimating sample purity. Samples with an A260/A280 ratio of ~1.8 typically provide optimal sequencing results. Send sample for sequencingSend samples to the Engencore sequencing facility at the University of South Carolina. Check their website for more specific details about what else to include in the shipment.Send samples overnight on dry ice PCR AmplificationPre-amplification checklist:All pipetting should be done with aerosol barrier, PCR certified pipet tips in a PCR work stationOn ice thaw primers, DNA and PCR master mixDecontaminate pipettors and interior surfaces of a PCR workstation with DNA/RNA Away or 10% bleach solution.UV irradiate PCR work station interior, pipettors and consumables for minimum 15 minAll samples should be amplified in triplicate.Include negative (no template) and positive (DNA isolated from mushroom) controls. Primer information:Forward primer:454 primer B + 'AG' linker + SSU817fGCCTTGCCAGCCCGCTCAGAGTTAGCATGGAATAATRRAATAGGA Reverse Primer:454 primer A + 12-base bar code + 'AC' linker + ssu1196rGCCTCCCTCGCGCCATCAG-12 base bar code-ACTCTGGACCTGGTGAGTTTCCExample barcode: ACACACTATGGCExample primer sequence: GCCTCCCTCGCGCCATCAGACACACTATGGCACTCTGGACCTGGTGAGTTTCC PCR Cocktail:22.5 ul PCR master mix (we use the Invitrogen Platinum PCR SuperMix)1 ul BSA0.75 ul 10 uM forward primer (all samples get the same forward primer)0.75 ul 10 uM reverse primer (all samples get a unique barcoded reverse primer)1 ul template DNA PCR cycle:Samples are initially denatured at 94°C for 10 min, then amplified using 30 cycles of 94°C for 45 sec, 52°C for 30 sec, and 72°C for 90 sec. A final extension of 10 min at 72°C is added at the end of the program to ensure complete amplification of the target region. Check for PCR product on a gel Amplicon CleaningCombine the triplicate PCR reactions into a single volumeClean PCR reactions with a PCR clean up kit. The MoBio UltraClean PCR clean up kit and the Invitrogen Purelink PCR purification kit have both worked well. Amplicon QuantificationUse the qubit system for quantifying DNA, with a few modifications.Instead of using the qubit assay tubes, prepare the samples in black microplates.Perform assay with 1 ul of PCR productRead the plates on the microplate reader in the Allison lab set at 485ex /530em. Estimate amplicon concentration using the standard curve. Amplicon PoolingCombine equal amounts of DNA per sample into single vessel (usually a 15 mL tube).Concentrate pooled amplicons using the Invitorgen Purelink PCR purification kitRun a gel to visualize pooled samplesDepending on how your pooled amplicons look on the gel, you may need to gel purify the pooled amplicons. Pyrosequencing is biased towards shorter reads. If you have a messy band on your gel, it is important to purify your samples to improve the quality of the DNA you will receive. We used the Qiagen QIAquick Gel Extraction Kit. We ran samples out on a 1.4 % gel. The main drawback to performing a gel extraction is that you will almost certainly lose some of your sample. You may need to pool additional PCR product and perform a second gel extraction to obtain enough sample for sequencing. Remove contaminants from gel purified samples using an Invitrogen Purelink PCR purification kit. Estimate final sample concentration and purityUse the qubit system for estimating sample concentration. The sequencing facility requires 2-3 ug of DNA for sequencing.Use the Nanodrop in the Martiny lab for estimating sample purity. Samples with an A260/A280 ratio of ~1.8 typically provide optimal sequencing results. Send sample for sequencingPre-amplification checklist:All pipetting should be done with aerosol barrier, PCR certified pipet tips in a PCR work stationOn ice thaw primers, DNA and PCR master mixDecontaminate pipettors and interior surfaces of a PCR workstation with DNA/RNA Away or 10% bleach solution.UV irradiate PCR work station interior, pipettors and consumables for minimum 15 minAll samples should be amplified in triplicate.Include negative (no template) and positive (DNA isolated from mushroom) controls. Primer information:Forward primer:454 primer B + 'AG' linker + SSU817fGCCTTGCCAGCCCGCTCAGAGTTAGCATGGAATAATRRAATAGGA Reverse Primer:454 primer A + 12-base bar code + 'AC' linker + ssu1196rGCCTCCCTCGCGCCATCAG-12 base bar code-ACTCTGGACCTGGTGAGTTTCCExample barcode: ACACACTATGGCExample primer sequence: GCCTCCCTCGCGCCATCAGACACACTATGGCACTCTGGACCTGGTGAGTTTCC PCR Cocktail:22.5 ul PCR master mix (we use the Invitrogen Platinum PCR SuperMix)1 ul BSA0.75 ul 10 uM forward primer (all samples get the same forward primer)0.75 ul 10 uM reverse primer (all samples get a unique barcoded reverse primer)1 ul template DNA PCR cycle:Samples are initially denatured at 94°C for 10 min, then amplified using 30 cycles of 94°C for 45 sec, 52°C for 30 sec, and 72°C for 90 sec. A final extension of 10 min at 72°C is added at the end of the program to ensure complete amplification of the target region. Check for PCR product on a gel Amplicon CleaningCombine the triplicate PCR reactions into a single volumeClean PCR reactions with a PCR clean up kit. The MoBio UltraClean PCR clean up kit and the Invitrogen Purelink PCR purification kit have both worked well. Amplicon QuantificationUse the qubit system for quantifying DNA, with a few modifications.Instead of using the qubit assay tubes, prepare the samples in black microplates.Perform assay with 1 ul of PCR productRead the plates on the microplate reader in the Allison lab set at 485ex /530em. Estimate amplicon concentration using the standard curve. Amplicon PoolingCombine equal amounts of DNA per sample into single vessel (usually a 15 mL tube).Concentrate pooled amplicons using the Invitorgen Purelink PCR purification kitRun a gel to visualize pooled samplesDepending on how your pooled amplicons look on the gel, you may need to gel purify the pooled amplicons. Pyrosequencing is biased towards shorter reads. If you have a messy band on your gel, it is important to purify your samples to improve the quality of the DNA you will receive. We used the Qiagen QIAquick Gel Extraction Kit. We ran samples out on a 1.4 % gel. The main drawback to performing a gel extraction is that you will almost certainly lose some of your sample. You may need to pool additional PCR product and perform a second gel extraction to obtain enough sample for sequencing. Remove contaminants from gel purified samples using an Invitrogen Purelink PCR purification kit. Estimate final sample concentration and purityUse the qubit system for estimating sample concentration. The sequencing facility requires 2-3 ug of DNA for sequencing.Use the Nanodrop in the Martiny lab for estimating sample purity. Samples with an A260/A280 ratio of ~1.8 typically provide optimal sequencing results. Send sample for sequencingSend samples to the Engencore sequencing facility at the University of South Carolina. Check their website for more specific details about what else to include in the shipment.Send samples overnight on dry icePrimer information:Forward primer:454 primer B + 'AG' linker + SSU817fGCCTTGCCAGCCCGCTCAGAGTTAGCATGGAATAATRRAATAGGAReverse Primer:454 primer A + 12-base bar code + 'AC' linker + ssu1196rGCCTCCCTCGCGCCATCAG-12 base bar code-ACTCTGGACCTGGTGAGTTTCCExample barcode: ACACACTATGGCExample primer sequence: GCCTCCCTCGCGCCATCAGACACACTATGGCACTCTGGACCTGGTGAGTTTCCPCR Cocktail:22.5 ul PCR master mix (we use the Invitrogen Platinum PCR SuperMix)1 ul BSA0.75 ul 10 uM forward primer (all samples get the same forward primer)0.75 ul 10 uM reverse primer (all samples get a unique barcoded reverse primer)1 ul template DNAPCR cycle:Samples are initially denatured at 94°C for 10 min, then amplified using 30 cycles of 94°C for 45 sec, 52°C for 30 sec, and 72°C for 90 sec. A final extension of 10 min at 72°C is added at the end of the program to ensure complete amplification of the target region. Check for PCR product on a gel Amplicon CleaningCombine the triplicate PCR reactions into a single volumeClean PCR reactions with a PCR clean up kit. The MoBio UltraClean PCR clean up kit and the Invitrogen Purelink PCR purification kit have both worked well. Amplicon QuantificationUse the qubit system for quantifying DNA, with a few modifications.Instead of using the qubit assay tubes, prepare the samples in black microplates.Perform assay with 1 ul of PCR productRead the plates on the microplate reader in the Allison lab set at 485ex /530em. Estimate amplicon concentration using the standard curve. Amplicon PoolingCombine equal amounts of DNA per sample into single vessel (usually a 15 mL tube).Concentrate pooled amplicons using the Invitorgen Purelink PCR purification kitRun a gel to visualize pooled samplesDepending on how your pooled amplicons look on the gel, you may need to gel purify the pooled amplicons. Pyrosequencing is biased towards shorter reads. If you have a messy band on your gel, it is important to purify your samples to improve the quality of the DNA you will receive. We used the Qiagen QIAquick Gel Extraction Kit. We ran samples out on a 1.4 % gel. The main drawback to performing a gel extraction is that you will almost certainly lose some of your sample. You may need to pool additional PCR product and perform a second gel extraction to obtain enough sample for sequencing. Remove contaminants from gel purified samples using an Invitrogen Purelink PCR purification kit. Estimate final sample concentration and purityUse the qubit system for estimating sample concentration. The sequencing facility requires 2-3 ug of DNA for sequencing.Use the Nanodrop in the Martiny lab for estimating sample purity. Samples with an A260/A280 ratio of ~1.8 typically provide optimal sequencing results. Send sample for sequencing Amplicon CleaningCombine the triplicate PCR reactions into a single volumeClean PCR reactions with a PCR clean up kit. The MoBio UltraClean PCR clean up kit and the Invitrogen Purelink PCR purification kit have both worked well. Amplicon QuantificationUse the qubit system for quantifying DNA, with a few modifications.Instead of using the qubit assay tubes, prepare the samples in black microplates.Perform assay with 1 ul of PCR productRead the plates on the microplate reader in the Allison lab set at 485ex /530em. Estimate amplicon concentration using the standard curve. Amplicon PoolingCombine equal amounts of DNA per sample into single vessel (usually a 15 mL tube).Concentrate pooled amplicons using the Invitorgen Purelink PCR purification kitRun a gel to visualize pooled samplesDepending on how your pooled amplicons look on the gel, you may need to gel purify the pooled amplicons. Pyrosequencing is biased towards shorter reads. If you have a messy band on your gel, it is important to purify your samples to improve the quality of the DNA you will receive. We used the Qiagen QIAquick Gel Extraction Kit. We ran samples out on a 1.4 % gel. The main drawback to performing a gel extraction is that you will almost certainly lose some of your sample. You may need to pool additional PCR product and perform a second gel extraction to obtain enough sample for sequencing. Remove contaminants from gel purified samples using an Invitrogen Purelink PCR purification kit. Estimate final sample concentration and purityUse the qubit system for estimating sample concentration. The sequencing facility requires 2-3 ug of DNA for sequencing.Use the Nanodrop in the Martiny lab for estimating sample purity. Samples with an A260/A280 ratio of ~1.8 typically provide optimal sequencing results. Send sample for sequencingCombine the triplicate PCR reactions into a single volumeClean PCR reactions with a PCR clean up kit. The MoBio UltraClean PCR clean up kit and the Invitrogen Purelink PCR purification kit have both worked well. Amplicon QuantificationUse the qubit system for quantifying DNA, with a few modifications.Instead of using the qubit assay tubes, prepare the samples in black microplates.Perform assay with 1 ul of PCR productRead the plates on the microplate reader in the Allison lab set at 485ex /530em. Estimate amplicon concentration using the standard curve. Amplicon PoolingCombine equal amounts of DNA per sample into single vessel (usually a 15 mL tube).Concentrate pooled amplicons using the Invitorgen Purelink PCR purification kitRun a gel to visualize pooled samplesDepending on how your pooled amplicons look on the gel, you may need to gel purify the pooled amplicons. Pyrosequencing is biased towards shorter reads. If you have a messy band on your gel, it is important to purify your samples to improve the quality of the DNA you will receive. We used the Qiagen QIAquick Gel Extraction Kit. We ran samples out on a 1.4 % gel. The main drawback to performing a gel extraction is that you will almost certainly lose some of your sample. You may need to pool additional PCR product and perform a second gel extraction to obtain enough sample for sequencing. Remove contaminants from gel purified samples using an Invitrogen Purelink PCR purification kit. Estimate final sample concentration and purityUse the qubit system for estimating sample concentration. The sequencing facility requires 2-3 ug of DNA for sequencing.Use the Nanodrop in the Martiny lab for estimating sample purity. Samples with an A260/A280 ratio of ~1.8 typically provide optimal sequencing results. Send sample for sequencingSend samples to the Engencore sequencing facility at the University of South Carolina. Check their website for more specific details about what else to include in the shipment.Send samples overnight on dry ice Amplicon QuantificationUse the qubit system for quantifying DNA, with a few modifications.Instead of using the qubit assay tubes, prepare the samples in black microplates.Perform assay with 1 ul of PCR productRead the plates on the microplate reader in the Allison lab set at 485ex /530em. Estimate amplicon concentration using the standard curve. Amplicon PoolingCombine equal amounts of DNA per sample into single vessel (usually a 15 mL tube).Concentrate pooled amplicons using the Invitorgen Purelink PCR purification kitRun a gel to visualize pooled samplesDepending on how your pooled amplicons look on the gel, you may need to gel purify the pooled amplicons. Pyrosequencing is biased towards shorter reads. If you have a messy band on your gel, it is important to purify your samples to improve the quality of the DNA you will receive. We used the Qiagen QIAquick Gel Extraction Kit. We ran samples out on a 1.4 % gel. The main drawback to performing a gel extraction is that you will almost certainly lose some of your sample. You may need to pool additional PCR product and perform a second gel extraction to obtain enough sample for sequencing. Remove contaminants from gel purified samples using an Invitrogen Purelink PCR purification kit. Estimate final sample concentration and purityUse the qubit system for estimating sample concentration. The sequencing facility requires 2-3 ug of DNA for sequencing.Use the Nanodrop in the Martiny lab for estimating sample purity. Samples with an A260/A280 ratio of ~1.8 typically provide optimal sequencing results. Send sample for sequencingUse the qubit system for quantifying DNA, with a few modifications.Instead of using the qubit assay tubes, prepare the samples in black microplates.Perform assay with 1 ul of PCR productRead the plates on the microplate reader in the Allison lab set at 485ex /530em. Estimate amplicon concentration using the standard curve. Amplicon PoolingCombine equal amounts of DNA per sample into single vessel (usually a 15 mL tube).Concentrate pooled amplicons using the Invitorgen Purelink PCR purification kitRun a gel to visualize pooled samplesDepending on how your pooled amplicons look on the gel, you may need to gel purify the pooled amplicons. Pyrosequencing is biased towards shorter reads. If you have a messy band on your gel, it is important to purify your samples to improve the quality of the DNA you will receive. We used the Qiagen QIAquick Gel Extraction Kit. We ran samples out on a 1.4 % gel. The main drawback to performing a gel extraction is that you will almost certainly lose some of your sample. You may need to pool additional PCR product and perform a second gel extraction to obtain enough sample for sequencing. Remove contaminants from gel purified samples using an Invitrogen Purelink PCR purification kit. Estimate final sample concentration and purityUse the qubit system for estimating sample concentration. The sequencing facility requires 2-3 ug of DNA for sequencing.Use the Nanodrop in the Martiny lab for estimating sample purity. Samples with an A260/A280 ratio of ~1.8 typically provide optimal sequencing results. Send sample for sequencingSend samples to the Engencore sequencing facility at the University of South Carolina. Check their website for more specific details about what else to include in the shipment.Send samples overnight on dry ice Amplicon PoolingCombine equal amounts of DNA per sample into single vessel (usually a 15 mL tube).Concentrate pooled amplicons using the Invitorgen Purelink PCR purification kitRun a gel to visualize pooled samplesDepending on how your pooled amplicons look on the gel, you may need to gel purify the pooled amplicons. Pyrosequencing is biased towards shorter reads. If you have a messy band on your gel, it is important to purify your samples to improve the quality of the DNA you will receive. We used the Qiagen QIAquick Gel Extraction Kit. We ran samples out on a 1.4 % gel. The main drawback to performing a gel extraction is that you will almost certainly lose some of your sample. You may need to pool additional PCR product and perform a second gel extraction to obtain enough sample for sequencing. Remove contaminants from gel purified samples using an Invitrogen Purelink PCR purification kit. Estimate final sample concentration and purityUse the qubit system for estimating sample concentration. The sequencing facility requires 2-3 ug of DNA for sequencing.Use the Nanodrop in the Martiny lab for estimating sample purity. Samples with an A260/A280 ratio of ~1.8 typically provide optimal sequencing results. Send sample for sequencingCombine equal amounts of DNA per sample into single vessel (usually a 15 mL tube).Concentrate pooled amplicons using the Invitorgen Purelink PCR purification kitRun a gel to visualize pooled samplesDepending on how your pooled amplicons look on the gel, you may need to gel purify the pooled amplicons. Pyrosequencing is biased towards shorter reads. If you have a messy band on your gel, it is important to purify your samples to improve the quality of the DNA you will receive. We used the Qiagen QIAquick Gel Extraction Kit. We ran samples out on a 1.4 % gel. The main drawback to performing a gel extraction is that you will almost certainly lose some of your sample. You may need to pool additional PCR product and perform a second gel extraction to obtain enough sample for sequencing. Remove contaminants from gel purified samples using an Invitrogen Purelink PCR purification kit. Estimate final sample concentration and purityUse the qubit system for estimating sample concentration. The sequencing facility requires 2-3 ug of DNA for sequencing.Use the Nanodrop in the Martiny lab for estimating sample purity. Samples with an A260/A280 ratio of ~1.8 typically provide optimal sequencing results. Send sample for sequencingSend samples to the Engencore sequencing facility at the University of South Carolina. Check their website for more specific details about what else to include in the shipment.Send samples overnight on dry ice Estimate final sample concentration and purityUse the qubit system for estimating sample concentration. The sequencing facility requires 2-3 ug of DNA for sequencing.Use the Nanodrop in the Martiny lab for estimating sample purity. Samples with an A260/A280 ratio of ~1.8 typically provide optimal sequencing results. Send sample for sequencingUse the qubit system for estimating sample concentration. The sequencing facility requires 2-3 ug of DNA for sequencing.Use the Nanodrop in the Martiny lab for estimating sample purity. Samples with an A260/A280 ratio of ~1.8 typically provide optimal sequencing results. Send sample for sequencingSend samples to the Engencore sequencing facility at the University of South Carolina. Check their website for more specific details about what else to include in the shipment.Send samples overnight on dry iceSend samples to the Engencore sequencing facility at the University of South Carolina. Check their website for more specific details about what else to include in the shipment.Send samples overnight on dry ice

Proper citation: Treseder K, Holden S, Maltz M 2017. Treseder Lab Pyrosequencing Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.jgacjse Copy   

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Authors: Yuan Yao

Proper citation: Yuan Yao 2018. Bacterial two hybrid analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.qaedsbe Copy   

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Authors: Lara Shepherd

Proper citation: Lara Shepherd 2019. ddRAD-Seq protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.zgyf3xw Copy   

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Authors: H. I. Cruz-Rosas, F. Riquelme, P. Santiago, L. Rendón, T. Buhse, F. Ortega-Gutiérrez, R. Borja-Urby, D. Mendoza, C. Gaona, P. Miramontes, G. Cocho.
Summary: SAMPLES. We used a meteorite specimen of 297.8 g in weight, register number IG-A7, from the Colección Nacional de Meteoritas of the Instituto de Geología, Universidad Nacional Autónoma de México (UNAM), Mexico City (figure). For transmission electron microscopy (TEM), a ~ 4 cm long fragment of the large specimen were freshly fractured and cut by using a diamond knife in a clean room. Later, smaller clean fragments (~ 50 mg in total) were extracted from the interior, avoiding contamination from the fusion crust or surface areas. The resulting crude sample were powdered in an agate mortar and suspended in high-purity ethanol to achieve homogeneous dispersion without agglomerates. For HRTEM analysis, a 5µl pipette was add a portion of that suspension to a lacey carbon grid coated with formvar. Before being used, the TEM grid was carefully inspected in order to rule out any possible contamination. HRTEM. High Resolution TEM imaging of chondrite samples was conducted using an Aberration Corrected Cold Field Emission Scanning Transmission Electron Microscope Jeol JEM-ARM200CF at the Centro de Nanociencias y Micro y Nanotecnologías, Instituto Politécnico Nacional, Mexico City. The TEM microscope is equipped with cold field emission gun, Cs-corrector, and high angle annular dark field (HAADF) detector and has ultra-high resolution of 0.72 Å. We utilized an electron beam spot with a condenser aperture of 60 nm at 200 kV for less than 30 seconds. Several locations on individual samples were analyzed. Fast Fourier Transform (FFT) analysis and image processing were applied using the freely available Digital Micrograph (GATAN) software attached at the microscope.

Proper citation: H. I. Cruz-Rosas, F. Riquelme, P. Santiago, L. Rendón, T. Buhse, F. Ortega-Gutiérrez, R. Borja-Urby, D. Mendoza, C. Gaona, P. Miramontes, G. Cocho. 2019. Allende HRTEM study. protocols.io https://dx.doi.org/10.17504/protocols.io.3f4gjqw Copy   

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Authors: Marda Jorgensen, Jerelyn Nick
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community
Summary: The purpose of this Standard Operating Procedure (SOP) is to outline procedures for the OCT embedding of HuBMAP frozen fixed specimens.

Proper citation: Marda Jorgensen, Jerelyn Nick 2020. HuBMAP: Embedding Fixed Frozen OCT Samples. protocols.io https://dx.doi.org/10.17504/protocols.io.basniede Copy   

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Authors: Kiichi Hirota, Yoshiyuki Matsuo

Proper citation: Kiichi Hirota, Yoshiyuki Matsuo 2018. Measurement of cellular oxygen consumption and extracellular acidification. protocols.io https://dx.doi.org/10.17504/protocols.io.v92e98e Copy   

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Authors: Grace Basa
Group: Neubig_Lab
Summary: This is the protocol for making and running a 12% SDS-PAGE Western blot. Protocol starts from pouring gels all the way to reading the membrane. 

Proper citation: Grace Basa 2018. 12% SDS-PAGE Western Blot . protocols.io https://dx.doi.org/10.17504/protocols.io.rwrd7d6 Copy   

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Authors: Saul Moore
Group: Behavioural Genomics

Proper citation: Saul Moore 2021. Making normal NGM for imaging plates (Cabreiro Lab). protocols.io https:// Copy   

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Authors: Mia Maltz, Allison SD, German D, Looby C.
Summary: We conducted fluorimetric assays for each of three hydrolytic enzymes: b-glucosidase and cellobiohydrolase, and N-acetyl-glucosaminidase in black microplates. We measured fluorescence at 365 nm excitation and 450 nm emission. From each sample, we recorded fluorescence values for MUB substrate (substrate control), homogenate (homogenate control), MUB standards in the presence of maleate buffer (standard), and MUB in the presence of homogenate. We calculated potential extracellular enzyme activity of NAG, BG, and CBH from each litter sample.  

Proper citation: Mia Maltz, Allison SD, German D, Looby C. 2017. Extracellular enzyme assays (NAG, BG, and CBH). protocols.io https://dx.doi.org/10.17504/protocols.io.jf8cjrw Copy   

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Authors: Kelsey Knight
Group: BioLegend

Proper citation: Kelsey Knight 2018. Sandwich ELISA Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.tm9ek96 Copy   

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Authors: Eric Carpenter
Group: GigaScience Press, BGI
Summary: Implemented by: Beijing Genomics InstituteThis protocol is part of a collection of eighteen protocols used to isolate total RNA from plant tissue. (RNA Isolation from Plant Tissue Collection: https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6)

Proper citation: Eric Carpenter 2019. RNA Isolation from Plant Tissue Protocol 5: pBIOZOL Method. protocols.io https://dx.doi.org/10.17504/protocols.io.4rdgv26 Copy   

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Authors: Eduardo Fleury
Summary: Objective: To evaluate the ability of BMRI to detect silicone gel bleeding in a prospective observational study including consecutive patients referred for a BMRI scan.Methods: From January 2017 until now, patients referred for BMRI were evaluated in a prospective observational study. Patients who had breast implants were included. BMRI recorded 9 findings according to BI-RADS lexicon and SIGBIC findings, considered equivocal features to detect gel bleeding (GB). Three new original imaging features were added for SIGBIC diagnosis: black drop signal; T2* hypersignal mass; and delayed contrast enhancement, considered as irrevocable signs. The presence of silicones corpuscles was confirmed by percutaneous biopsy or surgical capsulectomy. The accuracy of BMRI SIGBIC findings to predict GB was determined. We also used univariate analysis for the equivocal features for GB diagnosis. The Backward method was applied for a multivariate Logistic Regression model for the equivocal features.

Proper citation: Eduardo Fleury 2020. SILICONE INDUCED GRANULOMA OF BREAST IMPLANT CAPSULE. protocols.io https://dx.doi.org/10.17504/protocols.io.bfn4jmgw Copy   

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Authors: Aleksey Pichugin, Alexander Bagaev, Ravshan Ataullakhanov
Summary: ELISPOT allows detection of single IFN-γ-secreting cell, but the use of unseparated spleen cells removes the advantages of the method due to a high back ground of IFN-γ-production and uncertainty of the phenotype of secreting cells (CD4, CD8 or NK).We used high purity sorted CD4 and CD8 cells.For in vitro restimulation of CD8 T-cells, we used dendritic cells that present antigenic epitopes  of  M2,  NP  or  of  adenovirus  in  the  context  of  MHC  class  I.  In  this  instance,  dendritic  cells  were  transduced  with  Ad5-tet-M2,  Ad5-tet-NP  or  Ad5-null, respectively.For restimulation of CD4 T-cells in vitro, we used dendritic cells that present antigenic M2 or NP epitopes in the context of MHC class II. For this purpose, dendritic cells were additionally activated by lipopolysaccharide E. coli (1 μg/ml) and were loaded with recombinant protein NP or M2e peptide..

Proper citation: Aleksey Pichugin, Alexander Bagaev, Ravshan Ataullakhanov 2018. High-fidelity detection of IFN-? secreting CD4 and CD8 cells in response to soluble or Ad5 transduced influenza antigens using ELISPOT and cell sorting. protocols.io https://dx.doi.org/10.17504/protocols.io.jn4cmgw Copy   

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Authors: Haven Himmighoefer
Summary: Is used in a multitude of experiments, but is often used as an ingredient in 10x TBE buffer

Proper citation: Haven Himmighoefer 2017. 0.5 M EDTA (0.5 L). protocols.io https://dx.doi.org/10.17504/protocols.io.ks7cwhn Copy   

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Authors: James Thornton
Group: Hurwitz Lab, MetaFunc Course

Proper citation: James Thornton 2016. Installing Anvi'o using a Docker Image. protocols.io https://dx.doi.org/10.17504/protocols.io.gejbtcn Copy   

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Authors: Caio Maximino, Jeisiane Souza de Oliveira, Monica Gomes Lima-Maximino
Group: Fish behavior and physiology
Summary: Zebrafish, and other small teleosts, are used as experimental models to evaluate human pathologies, including those linked to oxidative and nitrosative stress, inflammation, and nitric oxide signaling. The protocol presents an optimized technique to quantify nitrite levels, in zebrafish tissues, focusing on the brain and head kidney. The protocol is based on the classical Griess diazotization reaction (Griess, 1858) method.

Proper citation: Caio Maximino, Jeisiane Souza de Oliveira, Monica Gomes Lima-Maximino 2018. Optimized protocol for quantification of nitrite levels in brain and head kidney tissue samples in adult zebrafish. protocols.io https://dx.doi.org/10.17504/protocols.io.sabeaan Copy   

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Authors: Chunwei Zeng, Guixue Hou
Group: Metabolomics Protocols & Workflows

Proper citation: Chunwei Zeng, Guixue Hou 2017. UPLC-MS/MS procedures of lipidomics for plasma. protocols.io https://dx.doi.org/10.17504/protocols.io.imncc5e Copy   

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