eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Four different samples were employed for experiments, designed as follows:</p>
<ol>
<li><u>HABP/HA</u>: cantilever functionalised with hyaluronic acid binding protein, untreated cell sample;</li>
<li><u>BSA/HA</u>: cantilever functionalised with bovine serum albumin, untreated cell sample;</li>
<li><u>untreated/HA</u>: non-functionalised cantilever, untreated cell sample;</li>
<li><u>HABP/HAase</u>: cantilever functionalised with hyaluronic acid binding protein, cell sample treated with HAase</li>
</ol>
<p> </p>
<p>The steps of activation are listed in this protocol and were the same for Sample 1 (HABP/HA, functionalisation molecule: hyaluronic acid binding protein), Sample 2 (BSA/HA, functionalisation molecule: bovine serum albumin) and Sample 4 (HABP/HAase, functionalisation molecule: hyaluronic acid binding protein). The cantilevers used to test cells in Sample 3 (untreated/HA) were not treated.</p>
<p> </p>
<p>Similar protocols were described in:</p>
<p>* Maki K et al. Mechano-adaptive sensory mechanism of α-catenin under tension. Sci Rep 2016;6: 24878. doi:10.1038/srep24878</p>
<p>* Maki K et al. Real-time TIRF observation of vinculin recruitment to stretched α-catenin by AFM. Sci Rep 2018;8: 1–8. doi:10.1038/s41598-018-20115-8</p>
<p> </p>

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: Prodentim Australia NZ or Canada Reviews
AUTHORS: (prodentimaustralia)

[DESCRIPTION]

Prodentim Australia helps reverse acidic processes that create optimal conditions for bacteria. This can help stop deadly bacteria in their tracks, after which the supplement can move on to stage two. The Prodentim Australia supplement can help slow down and prevent tooth decay. It consists of several minerals and nutrients that help revitalize teeth and prevent plaque formation.Therefore, it can help protect the gums of your teeth from harm and disease. 


Official Web Click Here :-

https://ipsnews.net/business/2022/07/21/prodentim-australia-reviews-2022-prodentim-nz-price-ingredients-scam-or-where-to-buy-pro-dentim-in-new-zealand-or-australia/

https://ipsnews.net/business/2022/07/19/prodentim-uk-reviews-scam-or-legit-ingredients-customer-real-reviews-price-or-buy-form-official-website-in-united-kingdom/

https://prodentiminca.cgsociety.org/ju33/prodentim-reviews-pr


[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is the protocol I have developed to genterate High'ish' Molecular Weight gDNA from gram negative bacterial speices to use for ONT MinION sequencing.</p>
<p> </p>
<p>This protocol was intially developed for the high throughput extraction of bacterial gDNA for Illumina short read sequencing. To perform our high throughput work we utislise Beckman Coulter Biomek liquid handling robots and the Beckman Coutler gDNA extraction kit Genfind v2. This kit utilises magnetic pariticles and is relatively gentle. We have found that gDNA extracted using this method has a high'ish' molecular weight. On a fragment analyser the GNDA is typically a clean single peak &gt;60 kb (the limits of the fragment analyser). ONT MinION libraries generated wiht this gDNA typically have N50s in excess of 20 kb and the data is suitable for the assembly automated of almost bacterial genomes we have tested to date. This protocol will not give you the ultra long 'whale' reads that others have reported but it is a good compromise between HMW DNA, speed and ease of use and capacity to scale up to high throughput. </p>
<p> </p>
<p><strong>NOTE</strong></p>
<p>We have found that gDNA extracted using this method often have underepresentation of smaller plamids (&lt;20kbp). We are not sure exactly why this happens but possible reason are; the circular replicons do not bind as readily to the magnetic particles and so are excluded from the extraction or the plamids remain circule and so are not availabe to adapter ligation in the library prep. I will upload an additonal protocol for gDNA extraaction that focuses on enrichment of plasmid sequences. </p>
<p> </p>
<p> </p>

[STEPS]
?. 
?. 
?. 
?.
TITLE: Amyloid dye-binding analysis of α-synuclein fibrils
AUTHORS: Arpine Sokratian

[DESCRIPTION]
This protocol describes the amyloid binding assay used to capture the conformational signatures in expanded cohort of a-synuclein fibrils. Specific amyloid dyes indicated in the protocol display a distinct binding affinity to a-synuclein fibril variants and expose no signal in presence of native or unfolded protein

[STEPS]
SECTION: Dye preparation
1. Prepare / thaw down the dye powder at  Room temperature 
  ,    ,   
  

SECTION: Dye preparation
2. Dissolve the dyes in DMSO to final concentration 15 millimolar (mM)  for NIAD-4, Nile Red and FSB, 
Dissolve ThT in ddH2O to final concentration1 millimolar (mM) 
Store aliquots at -80 °C

SECTION: Dye preparation
3. Follow the protocol to prepare the batch of sonicated fibrils. If the samples have been prepared earlier and kept at -80, thaw down in a water bath. Then, perform a quality control check to ensure the size distribution and concentration are corresponding to values outlined in the protocol attached.
 


SECTION: Dye preparation
4. Evaluated sonicated fibrils should be diluted in PBS to concentration of 2 mg/mL. 
Prepare serial dilutions with dilution factor of 2 in PBS.
SECTION: Dye preparation
5. Once dilutions of fibrils are prepared, thaw down the frozen aliquots of amyloid dyes in water-bath set at 42 °C

SECTION: Dye preparation
6. Dilute amyloid dyes in PBS to 100 uM concentration (volume 2-3 mL in 15 mL falcon tubes). 
IMPORTANT: Dyes need to be diluted after all other reagents are ready
SECTION: Dye preparation
7. Add 5 ul of dyes for each replicate/ dilution accounted for  
IMPORTANT: Spin down the covered 384-well plate loaded with the dyes before moving on to the next step. 
SECTION: Dye preparation
8. Once 5 uL of diluted dyes have been added to the 384-well plate, add the fibril/protein to the dye-preloaded wells. As a standard curve, we optionally use BSA fibrils or standard recombinant sonicated a-synuclein fibrils (starting from 5 mg/mL concentration)
SECTION: Dye preparation
9. Cover the plate with a sealing foil. Spin down the plate at 500g for 30 sec. 
SECTION: Dye preparation
10. Set up the program in 
 

SECTION: Dye preparation
11. The program should include the incubation period of 5 min at continuous shaking at 200 rpm. Fluorescence intensity is recorded with excitation/emission spectra set at 468-15/510-20 for FBS, 535-20/585-30 for nile red and 448-10/482-10 nm for ThT. Monomeric mouse and human a-synuclein monomers should be used as a controls (include blank well with PBS as well). 
SECTION: Dye preparation
12. Extrapolate the results by subtracting the average blank value. Then, correlate these adjusted results with the standard curve. Ensure that the extrapolated values fall within the appropriate range of the standard curve.
TITLE: Media and reagents for Seminavis robusta cultivation and experiments
AUTHORS: Lev Tsypin, Aaron Turkewitz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes the culturing conditions used to grow </span><span style = "font-style:italic;">S. robusta </span><span>in the lab for all experiments.</span></div></div>

[STEPS]
?. [Preparation of F/2 medium]
Dissolve  in
[Tropic-Marin Bioactif (TMB)]
[MilliQ water]
?. [Preparation of F/2 medium]
Add
[sodium bicarbonate]
?. [Preparation of F/2 medium]
Autoclave
?. [Maintenance antibiotics (200x stock)]
Dissolve , , , and  in a final volume of .
[penicillin G sodium salt]
[ampicillin sodium salt]
[streptomycin sulfate]
[gentamycin sulfate]
[F/2 medium]
?. [Preparation of F/2 medium]
After cooling, add
[50x Guillard's supplement]
?. [Preparation of F/2 medium]
Filter using Stericup into pre-sterilized bottles
?. [Preparation of F/2 medium]
Store at 4C in the dark
?. [Maintenance antibiotics (200x stock)]
Aliquot and store at -20 C.
?. [Agar plates]
Dissolve  in
[TMB]
[MilliQ]
?. [Agar plates]
Add
[sodium bicarbonate]
?. [Agar plates]
Add , dissolve while stirring and heating (may require boiling)
0 µl
[agar]
?. [Agar plates]
Autoclave
?. [Agar plates]
After cooling below 60 C, add , as well as selective antibiotics, if desired.
[50x Guillard's supplement]
?. [Agar plates]
Pour approx.  per plate
19 ml
?. [Agar plates]
Store in the dark at 4 C
?. [Electroporation buffers]
in MilliQ,  in MilliQ, or  with   in MilliQ.
[sorbitol]
[mannitol]
[mannitol]
[F/2]
TITLE: Implementing a Health Curriculum for the Kupuna Class of Honolulu to Improve Health Literacy: An Evidence-Based Review
AUTHORS: Jaymie Barnes, Rayson-Lee Egan, hannabs, Louis Langi, pakjenna, cmh, Samia Valeria Ozorio Dutra

[DESCRIPTION]
The absence of specialized health
education for seniors may lead to decreased health literacy, increased health
risks, social isolation, and a strain on the healthcare system. Establishing a
health education class could address these issues, improve health outcomes, and
reduce preventable health problems. The review aims to assess the
implementation of a health promotion program to improve the health literacy of
elderly individuals.

*This protocol follows PRISMA-P guidelines and is provided as an attachment.* 

[STEPS]
TITLE: Protocol: Comparative efficacy of sedation or analgesia methods for reduction of anterior shoulder dislocation: a systematic review and network meta-analysis
AUTHORS: Minoru  Hayashi, Kenichi Kano, Naoto Kuroda, Norio Yamamoto, Akihiro Shiroshita, Yuki Kataoka

[DESCRIPTION]
The objectives are as follows:
To review systematically comparative efficacy of sedation or analgesia methods for reduction of anterior shoulder dislocation.

[STEPS]
TITLE: Desalting of Peptides to Prepare for Mass Spectrometry Analysis
AUTHORS: Alexandra Naba, jconsi

[DESCRIPTION]
Prior to proteomic analysis, peptide samples are desalted and eluted with freshly prepared 50% acetonitrile, 0.1% trifluoroacetic acid, followed by concentration in a vacuum concentrator. Peptides are then resuspended in freshly prepared 5% acetonitrile, 0.1% formic acid. 



 


[STEPS]
SECTION: Column Preparation
1. Column Preparation
SECTION: Column Preparation
1.1. Take a Pierce peptide desalting spin column and remove the white tip (do not remove the screw cap of the tube). Place in a 2mL tube and spin column at   for 1 min. 

SECTION: Column Preparation
1.2. Add 300 µLof acetonitrile. Spin at  for 1 minand discard flow-through.

SECTION: Column Preparation
1.3. Repeat this step once


 

SECTION: Sample Loading
2. Sample Loading 
SECTION: Sample Loading
2.1. Place the spin column in a new low-retention 2 mL tube labeled "flowthrough".

SECTION: Sample Loading
2.2. Load 300 µL of peptide sample into the tube and spin at   for 1 min. 
 


SECTION: Sample Loading
2.3. Based on the total sample volume, if:

2.3a) More than 300 µL of sample -> place into a new 2 mL tube and load the remaining volume
2.3b)Less than 300 µL -> reload the flow-through.



SECTION: Wash
3. Wash Sample
SECTION: Wash
3.1. Place the spin column in a new low-retention 2mL-tube and load300 µLof 0.1% TFA in HPLC-grade H2O. Centrifuge at3000 x g for  1 min.  Discard wash flow-through.

SECTION: Wash
3.2. Repeat step 3.1 2 more times.
 

SECTION: Sample Elution
4. Elute Samples
SECTION: Sample Elution
4.1. Place the spin column in a new 2mL low-retention tube labeled with the sample name.
SECTION: Sample Elution
4.2. Load 300 µLof 0.1% TFA, 50% acetonitrile in HPLC-grade H2O . Spin at  for 1 min. 


 


SECTION: Sample Elution
4.3. Transfer the spin column to another 2mL-low retention tube and repeat the step. 
SECTION: Sample Elution
4.4. Pool the two elution samples from 4.2 and 4.3. These are the desalted peptides.
SECTION: Sample Elution
4.5. Store at-20 °C.

SECTION: Lyophilization and Reconstitution
5. Lyophilization
SECTION: Lyophilization and Reconstitution
5.1. To remove reagents incompatible with mass spectrometry place tubes in SpeedVac™ until completely dry.
SECTION: Lyophilization and Reconstitution
5.2. Depending on the size of the peptide pellet, resuspend samples with20 -75 µLof 0.1% formic acid, 5% acetonitrile in HPLC-grade H2O.

SECTION: Lyophilization and Reconstitution
5.3. Vortex until completely resuspended.
SECTION: Sample Loading
2.4. Spin the sample at   for 1 min. Store “FT” at-80 °C for troubleshooting purpose. 
SECTION: Lyophilization and Reconstitution
5.4. Peptide concentration can be measures using a spectrophotometer or using peptide concentration measurement kits such as:   )
TITLE: P21.- REVISIÓN Y CONTROL DEL PROCESO DE PRESENTACIÓN PLANES DE INVESTIGACIÓN
AUTHORS: cgarcia

[DESCRIPTION]
Atendiendo a las particularidades de la Aplicación LAUREA ACADEMIC, el proceso de presentación del Plan de Investigación inicial por parte de los doctorandos, actualmente queda únicamente bajo la supervisión de tutores y directores. 

Desde la Escuela de Doctorado, creemos que este trámite es fundamental para el desarrollo del Programa y por este motivo facilitaremos a las Comisiones Académicas los mecanismos de control que aseguren que este proceso se realiza correctamente.

[STEPS]
SECTION: P21.- REVISIÓN Y CONTROL DEL PROCESO DE PRESENTACIÓN PLANES DE INVESTIGACIÓN
1. DESCRIPCIÓN DEL PROCESO

1.- En la primera semana de cada mes, la Secretaría de la EIDUCAM enviará un informe de estado de inscripciones de Planes de Investigación a las Comisiones Académicas. En este informe incluirá la siguiente información:

ALUMNOS PENDIENTES DE INSCRIBIR PLAN DE INVESTIGACIÓN
DNI, NOMBRE Y APELLIDOS, TUTOR, DIRECTOR, ESTADO 

 ALUMNOS QUE HAN REALIZADO CORRECTAMENTE LA INSCRIPCIÓN DEL PLAN DE  INVESTIGACIÓN
DNI, NOMBRE Y APELLIDOS, TUTOR, DIRECTOR, FECHA APROBACIÓN. OBSERVACIONES

Se considerará inscrito el Plan cuando la aprobación se haya realizado por parte del tutor o cualquiera de sus directores, se hará constar en observaciones quién ha realizado la aprobación y quién está pendiente de realizarla.
SECTION: P21.- REVISIÓN Y CONTROL DEL PROCESO DE PRESENTACIÓN PLANES DE INVESTIGACIÓN
2. 2.- Mensualmente, las Comisiones deberán remitir el informe recibido, resolviendo la aprobación definitiva de los planes de investigación o realizando las observaciones necesarias para que sean trasladadas a alumnos, tutor y director/es. Los informes de resolución de planes deben ser entregados a la Secretaría de la EIDUCAM, antes de la finalización del mes en curso.
SECTION: P21.- REVISIÓN Y CONTROL DEL PROCESO DE PRESENTACIÓN PLANES DE INVESTIGACIÓN
3. 3.- La Secretaría de la EIDUCAM, procederá al registro de planes aprobados.
TITLE: 4% PFA + 2.5% Glutaraldehyde Fixative
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to prepare 4% Paraformaldehyde (PFA) + 2.5% Glutaraldehyde Fixative. This is utilized to fix the tissue after electrophysiology recordings. </div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE: Immunophenotyping  of the peripheral blood immune cells by flow cytometry
AUTHORS: mariangela.massarocenere, Valerio Chiurchiù

[DESCRIPTION]
Protocol for immunophenotyping peripheral blood immune cells 

[STEPS]
1. Deeply anesthetize the animal. Check for no reflexes and slower breathing
2. Cut the animal's thorax and collect peripheral blood from the left ventricle
3. Lyse the whole blood with the RBC (1X) solution for 10 minutes at 37°C.
3.1. Upon lysis, wash cells and resuspend in cold PBS for immunophenotyping
4. Stain cells surface with FITC-conjugated anti-CD4(1:100, Biolegend), anti-agranulocytes (1:100, Miltenyi biotec), PerCP5.5-conjugated anti-CD8 (1:100, Biolegend) or anti-CD45 (1:100, Biolegend), APC-conjugated anti-CD25 (1:80, Biolegend), PE-conjugated anti-CD45RA (1:100, Biolegend), APC-conjugated anti- CD161 (1:100, Biolegend), PE-Vio770 conjugated anti-CD3 (1:100, REAffinity Miltenyi biotec) and APC-Vio770-conjugated anti-CD11b/c (1:100, REAffinity Miltenyi biotec.) for 15 minutes at 4°C in the dark
4.1. Wash 5 minutes in cold PBS
5. Incubate with a fixing solution (1:3) for 15 minutes at room temperature in the dark
5.1. Wash 5 minutes in cold PBS
6. Permeabilize cells with Cytofix/Cytoperm (1:10, Biolegend) and then stain them intracellularly with PE-CF594-conjugated anti-FoxP3 (1:50, BD Biosciences) in Cytoperm at room temperature for 30 min
7.  Acquire cells with an appropriate flow cytometer
8. Gate cells on CD45 to identify total leucocytes. Inside this gate, identify granulocytes with their specific marker, monocytes, and myeloid cells as CD11b/c+, T-lymphocytes as CD3+, B-lymphocytes as CD45RA+, and NK cells as CD161+ cells.
9. For each analysis, acquire at least 0.5x10^6 live by gating on aqua Live/Dead negative cells and then analyze using Flowjo software (Tree Star)
TITLE: Extraction of gDNA from Synechocystis 6803
AUTHORS: Dennis Dienst

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. [Cell harvest & disruption]
[4000 g]
?. Resuspend pellet in 10 mL TE bufferrepeat centrifugation (step 1)
?. 
?. Resuspend pellet in 1 mL TES bufferfreeze in liquid nitrogen
wear goggles when working with liquid nitrogenhttps://en.wikipedia.org/wiki/Liquid_nitrogen
?. incubate for 1h at 60°C under gentle agitationalternatively: 16h (o/n) at 37 °C
60 °C
?. [Phenol-chloroform extraction]
add 1 Vol.add 1 Vol. Phenol/Chloroform/Isoamyl alcohol (25:24:1) and mixcentrifuge for 10 min at 12000 g and 4°C
[12000 g]
4 °C
?. transfer upper (aqueous) phase to fresh 1.5 mL tubeadd 1 Vol. Phenol/Chloroform/Isoamyl alcohol (25:24:1) and mixcentrifuge for 10 min at 12000 g and 4°C
[12000 g]
4 °C
?. 
?. [DNA precipitation]
transfer upper (aqueous) phase to fresh 1.5 mL tubeadd 0.7 Vol. 2-propanol (isopropanol)incubate for 5 min at room temperature
[room temperature]
?. [12000 g]
4 °C
?. carefully remove 2-propanol add 300 µL 70% EtOHair-dry pellet for 1h at room temperature(i.e. leave tube open on your bench and enjoy a coffee or two)
DNA pellet might be hardly visible
[room temperature]
?. 
?. [optional: RNA removal]
add 2µL RNase A incubate 30 min at 37 °Cincubate 10 min at 72 °C
?. [Preparation of buffer solutions]
TE buffer:                                     10 mM Tris-HCl                                1 mM EDTA ; pH 8.0                           TES buffer:                                  50 mM Tris-HCl 1 mM EDTA, pH 8.025% (w/v) Saccharose
?. [optional: RNA removal]
Store DNA at -20 °C
?. add 5 mg × mL -1 (spatula tip) lysozyme, 3.2 units proteinase K and 100 µL 20% SDSincubate for 1h at 37 °C
37 °C
TITLE: Feeding of cats with hyper-immune eggs: an oral immunization protocol
AUTHORS: Angel  Justiz-Vaillant

[STEPS]
?. The anti-gp120 positive eggs (hyper-immune eggs) are fed to 3 out of 5 cats. Three  (3) adult cats, 2-3 years old (1 male and 4 female). A sixth cat that was a part of the control group died during the experiment.
?. Each cat receives on average of 2 eggs diluted in 5 volumes of soya milk weekly for 10 weeks.
?. Of the 5 cats used in the study, 3 are fed hyper-immune eggs (anti-gp120), while the remaining 2 cats are fed eggs from non-immunized chickens.
?. And these two cats that are fed with eggs from non immunized chickens are use as controls.
?. Blood samples (2 ml) are collected from each cat, after completion of the feeding.
?. They are all tested for anti-HIV antibodies by an indirect HIV ELISA.
TITLE: U54 SCENT 10X Genomics Visium Protocol 
AUTHORS: Karen Abramson, Simon Gregory

[DESCRIPTION]
This document outlines the 10X Genomics Visium Protocol used for normal lung and colon tissues for SCENT at Duke University. 

[STEPS]
SECTION: 10X Genomics Visium Protocol
1. Once slides received, the following 10x Genomics protocol is followed: 

https://cdn.10xgenomics.com/image/upload/v1660261285/support-documents/CG000409_Demonstrated_Protocol_VisiumSpatialFFPE_Deparaffin_H_E_RevC.pdf

https://cdn.10xgenomics.com/image/upload/v1660261285/support-documents/CG000407_VisiumSpatialGeneExpressionforFFPE_UserGuide_RevD.pdf
SECTION: 10X Genomics Visium Protocol
2. 10x Genomics kit is used – Visium (v1) Spatial for FFPE Gene Expression Kit, Human Transcriptome.
SECTION: 10X Genomics Visium Protocol
3. Libraries sequenced on Illumina sequencers to a read depth of a least 25k reads/spot, with read lengths of 28x10x10x50.
TITLE: General Taq PCR Master Mix -- CHEM 384/584
AUTHORS: Ken   Christensen

[DESCRIPTION]
SapphireAmp Fast PCR Master Mix contains a hot start PCR enzyme, optimized buffer, dNTP mixture, gel loading dye (blue), and a density reagent as a 2X premix. SapphireAmp Fast PCR Master Mix is optimized for fast PCR and offers a rapid extension rate (10 sec. per kb). The inclusion of blue dye and a density reagent allows direct loading of PCR products on an agarose gel for electrophoresis. The master mix format simplifies workflows and sample handling; simply add primers, template, and water and then begin PCR. SapphireAmp Fast PCR Master Mix is ideal for fast colony PCR screening. Fast colony PCR amplification of a 5 kb insert can be completed in approximately 1 hr 15 min. Furthermore, it is possible to amplify fragments up to 6 kb from genomic DNA templates.

[GUIDELINES]
Storage: -20℃ for long-term storage. 4℃ for short-term storage (up to 3 months). (Note) If used frequently, store at 4℃ ; the activity of the Master Mix may decrease with repeated freezing and thawing. Gently mix well before use and centrifuge briefly.

Application: 
・DNA amplification by PCR 
・Colony PCR

PCR Products: Since most PCR products amplified with SapphireAmp Fast PCR Master Mix have an A overhang added at 3’-termini, the obtained PCR product can be used directly for cloning into a T-vector. Additionally, it is possible to clone the product in a blunt-end vector after blunting and phosphorylation of the end. 

Dye Migration During Electrophoresis: When 5 μl of the PCR sample is loaded on a 1% gel made with Agarose L03 [TAKARA] (Cat. #5003) and subjected to electrophoresis, the blue dye fronts are detected at positions corresponding to 1 kb and 3 - 5 kb. The absorption maxima for the dyes are ～ 260 nm and 620 nm, respectively. The dyes may be removed by isolating and purifying the DNA fragment from the gel or extracting DNA with NucleoSpin Gel and PCR Clean-Up (Cat. #740609.50/.250), if necessary

[STEPS]
SECTION: Setup Reaction
1. To a 25 µL aliquot of a 2X Taq PCR Master Mix (e.g. TaqDog, or Sapphire Amp), add template (10-20 µl cleared lysate for colony PCR or  20-50 ng of purified DNA for typical PCR), forward and reverse primers to a final concentration of 200 nanomolar (nM). Adjust final volume to 50 µL with nuclease free water or autoclaved water.


 


SECTION: Run Reaction
2. followed by 30 cycles of 98°C, 5 sec; 55°C, 5 sec; and 72°C, 40 sec.