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

[DESCRIPTION]
How to run Prodigal version 2.6.3 (Hyatt et al. 2010) through the iMicrobe plaform.
 
Prodigal is a protein-coding gene prediction software tool for bacterial and archaeal genomes. Prodigal runs smoothly on finished genomes, draft genomes, and metagenomes. Please note that Prodigal has not been tested on viruses, and that Prodigal contains no special rules or routines to handle viral genomes.
 
More informations about Prodigal can be found here : https://github.com/hyattpd/Prodigal/wiki

[STEPS]
SECTION: Run prodigal on a single genome
?. 
SECTION: Run prodigal on an assembly
?.
TITLE: U Michigan - Nerve Conduction Velocity
AUTHORS: Eva Feldman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">To confirm the presence of diabetic neuropathy, nerve conduction velocity (NCV) studies are performed.  The animals are anesthetized with isoflurane to prevent discomfort.  Body temperature is monitered with a dermal temperature probe and maintained at 32ºC with a warming lamp during NCV.  Body temperature is maintained at 37ºC after NCV using a warming pad to ease animal stress from anesthetic.  The nerve studies last less than 30 min per rat or mouse. The electrodes are cleaned with 70% alcohol between animals to maintain pathogen-free status.</div></div>

[STEPS]
?. [Settings]
Motor tests o Duration .02 ms o Range 25 mA o low frequency filter 1 Hz o High frequency filter 10 kHz o Sensitivity 1 mV o Time 2 ms/div Sensory test o Duration .02 ms o Range 25 mA o low frequency filter 1 Hz o High frequency filter 10 kHz o Sensitivity 50 μV o Time 2 ms/div
?. [Procedure:]
To confirm the presence of diabetic neuropathy, nerve conduction velocity (NCV) studies are performed.  The animals will be anesthetized with isoflurane.  Body temperature is monitored with a dermal temperature probe and maintained at 34º C with a warming lamp during NCV.  Skin temperature is maintained at 32ºC during NCV using an infrared thermometer.  Body temperature is maintained at 34ºC after NCV using a warming pad to ease animal stress from anesthetic.  The nerve studies will last less than 15 min per rat or mouse. The electrodes are cleaned with 70% alcohol between animals to maintain pathogen-free status.Sciatic-tibial motor NCV is determined by stimulating distally at the sciatic notch and distally at the ankle via bipolar electrodes with supramaximal stimulation. The conduction velocity is calculated using two the points of stimulation along the nerve and measuring the resultant onset latency and distance.Sural Sensory NCV is determined by stimulating the sural nerve distally at the ankle via bipolar electrodes with supramaximal stimulation and recording at the fourth and fifth digit.   The conduction velocity is calculated using the onset latency and distance.Tail motor latency is determined by stimulating distally along the tail at a recorded distance of 3 cm.  The onset latency is used for the latency measurement.Tail sensory NCV is determined by stimulating proximally along the tail at a recorded distance of 3 cm.  The conduction velocity is calculated using the onset latency and distance.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Note: If the percentage of CD45+ cells in your sample is less than 50%, please follow Protocol A. If it is higher than 50% then please follow protocol B.</strong></p>
<p> </p>
<p>The cells targeted by the Nanobeads are either selected or depleted by incubating your sample with the directly conjugated magnetic particles. The magnetically labeled fraction is retained by the use of a magnetic separator. After collection of the targeted cells, downstream applications include functional assays, gene expression, phenotypic characterization, etc.<br /><br /></p>

[GUIDELINES]
<p>This procedure is optimized for the isolation of 10<sup>7</sup> to 2 x 10<sup>8</sup> cells per tube. If working with fewer than 10<sup>7</sup> cells, keep volumes as indicated for 10<sup>7</sup> cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water.</p>
<p> </p>
<p><strong>Reagents and instruments required:</strong></p>
<p> </p>
<p>-MojoSort™ Buffer (5X) (Cat. No. 480017)<br />-MojoSort™ Magnet (Cat. No. 480019) or compatible magnetic separation system<br />-Adjustable pipettes<br />-70 μm filters (one per sample)<br />-5 mL (12 x 75 mm) polystyrene tubes<br />-Reagents for sample preparation<br />-Reagents and instruments (Flow cytometer) to determine yield and purity</p>

[STEPS]
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TITLE: U Michigan - Massons Trichrome staining
AUTHORS: Jeff Hodgin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Massons Trichrome staining is used for detection of collagen fibers for tubulointerstitial fibrosis on formalin-fixed paraffin-embedded kidney tissue section. The collagen fibers will be stained blue, the nuclei black, and the background is stained red.</div></div>

[STEPS]
?. Wash 2x for 5 minutes in Xylene
?. Wash 2x for 3 minutes in 100% EtOH
?. Wash 1x for 2 minutes in 95% EtOH
?. Wash 1x for 1 minutes in 70% EtOH
?. Wash in running tap water for 3 minutes
?. Place in Bouins for one hour in 55 -56 degree oven or overnight at room temperature if needed.
?. Wash well in running water for 5-10 minutes or until yellow is gone.
?. Stain in Weigerts Hematoxylin for 10-20 minutes (usually 10 minutes).
?. Wash well in running water for 10 minutes.
?. Rinse distilled water.
?. Place slides in Biebrich Scarlet-Acid Fuchsin for 5-10 minutes.
?. Rinse in distilled water.
?. Place slides in the Phosphomolybdic-Phosphotungstic Acid Solution in a plastic coplin jar for 10-15 minutes or until collagen fibers are not red.
?. Place slides in Aniline Blue solution for 10 minutes.
?. Rinse in distilled water.
?. Place in 1% Acetic acid for 3 minutes.
?. Wash 2x for 3 minutes in 100% EtOH
?. Wash 1x for 2 minutes in 95% EtOH
?. Wash 2x for 5 minutes in Xylene
?. Mount slides with mounting medium- 1 drop
?. Insert Coverslip carefully, avoid bubble
TITLE: Size exclusion chromatography of cell lysates containing Tau aggregates
AUTHORS: Patricia Yuste Checa, Itika Saha, F. Ulrich Hartl, Mark S. Hipp

[DESCRIPTION]
This protocolcan be used to fractionate Tau aggregates from cell lysates by size exclusion chromatography. The protocol was optimized using HEK293 cells stably expressing and propagating aggregates of Tau repeat domain, containing the disease-related mutations P301L and V337M, fused to YFP (Clone 10 from Sanders et al., Neuron, 2014). However, any cell line containing Tau aggregates can be used.

[STEPS]
1. Lyse cell pellets with Triton buffer, Complete EDTA-free protease inhibitor cocktail (Roche) and benzonase for 20 min on ice. NOTE: Triton buffer: 0.05% Triton X-100/PBS or 1% Triton X-100/PBS (more efficient lysis) can be used to lyse cell pellets.
2. Clarify the lysates by centrifugation at 1,000 x g for 5 min at 4 °C and filter the supernatant with a PVDF 0.22 μm filter (Millex, #SLGVX13NL).
3. Quantify total protein by Bio-Rad Protein Assay (Bio-Rad) or Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).
4. Load 3 mg total protein on a Superose 6 HR10/30 (GE 875 Healthcare) column previously equilibrated with PBS. 
5. Analyze and quantify individual fractions by Western blot. NOTES: Use 15 µl of each fraction to detect TauRD-YFP by immunoblotting with anti-GFP antibody (Roche #11814460001).
6. The corresponding fractions, HMW and LMW species, can be pooled and quantified (using purified TauRD-YFP as standard) by immunoblotting to perform other biochemical assays or cell-based Tau seeding assays. 
 

5.1.   

4.1. NOTES: Cells from a confluent T75 flask lysed with 800 µL lysis buffer results in ~ 8-10 mg/mL total protein. Superose 6 HR10/30 (GE Healthcare): Flow rate 0.5 mL/min; 1mL fractions up to void volume of the column and 0.5 mL fractions from void volume to the end.
TITLE: Cylinder test in rats
AUTHORS: eduard.bentea, María Sanchiz Calvo, Veerle Baekelandt

[DESCRIPTION]
Protocol for performing the cylinder test in rats. The cylinder test evaluates asymmetry in forelimb use, and can be used in animal models with unilateral lesions of the nigrostriatal dopaminergic pathway.

[STEPS]
SECTION: Test
1. Bring cages to the behavioral room for at least 60 min prior to the test for habituation

SECTION: Test
2. Place each rat in a glass cylinder (20-cm wide), surrounded by mirrors to allow a full 360 degree view. Videotape from the front for 5 min or until the rat performs a minimum of 20 weight-bearing forepaw contacts with the cylinder walls.

SECTION: Test
3. Perform blinded analysis from the recorded videos of the number of contralateral forepaw contacts, and express these as a percentage of total forepaw contacts.
TITLE: First Strand Synthesis with Reverse Transcriptase (M0253)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
This protocol is for First Strand Synthesis with M-MuLV Reverse Transcriptase (M0253).

[BEFORE_START]
Prepare the following solutions:

10X RT buffer: 
      Tris-HCl (pH 8.3 @   25°C)  500 mM    KCl  750 mM    MgCl2  30 mM    DTT  100 mM  
 
dNTP mix (2.5 mM each in water titrated by Tris-HCl to pH 7.0)

[STEPS]
1. In a sterile microfuge tube add the following:  
 

2. Heat for 3-5 minutes at 65 °C-80 °C.
3. Spin briefly and place promptly on ice.
4. Add:
2 µL 
1 µL 
1 µL 

Final volume: 20 µL

5. Incubate at 42 °C for 60 min.
6. Inactivate enzyme at 90 °C for 10 min.
7. Store products at -20 °C or proceed to next step(s).
TITLE: 4% Paraformaldehye in .1M PB preparation
AUTHORS: Monique Copeland

[DESCRIPTION]
For perfusion solution.

[STEPS]
SECTION: Paraformaldehyde prep
1. Add 400 mL of MilliQ water to a 1000 mL 

 

SECTION: Paraformaldehyde prep
2. Add stir bar in beaker and set on hot plate at 50 °C  

SECTION: Paraformaldehyde prep
3. Add 5 drops (500ul) of 10N NaOH (from 60ml dropper)

 
(this makes the solution basic to help dissolve the paraformaldehyde)
SECTION: Paraformaldehyde prep
4. Weigh 20 g of Paraformaldehyde and add to MilliQ water.
(Stir about 60 min  or until all parafromaldehyde is dissolved and solution is clear)

 

SECTION: Paraformaldehyde prep
5. Make 1 Molarity (M) Phosphate buffer at PH 7.4

SECTION: Paraformaldehyde prep
7. Top off to 500ml with MilliQ water.
SECTION: Paraformaldehyde prep
8. Filter solution through .2um CA member filter.


 


SECTION: Paraformaldehyde prep
9. Lable container with '4% paraformaldehyed in .1M PB' and date. Store at 4 °C 
(Can also be aliqouted into 
 
and stored at -20 °C  for 3 months.)


SECTION: Paraformaldehyde prep
5.1. Prepare 1 Molarity (M) Soduim Phosphate Dibasic Dihydrate (Store stock at Room temperature .)



 



SECTION: Paraformaldehyde prep
5.2. Prepare 1 Molarity (M) Sodium Phospate Monobasic Anyhydrous (store stock at Room temperature )

 



SECTION: Paraformaldehyde prep
5.3. To make 100 mL add 19 mL  plus 81 mL  (store as stock at Room temperature )




SECTION: Paraformaldehyde prep
5.4. Add 50 mL to paraformaldehyde solution.

SECTION: Paraformaldehyde prep
6. Test PH and adjust solution to final desired pH using HCl or NaOH.
TITLE: Beam test
AUTHORS: Marina Lorente Picón, Núria Peñuelas, Ariadna Laguna, Miquel Vila

[DESCRIPTION]
Beam test for mice

[STEPS]
1. Mice are placed at the beginning of an elevated horizontal plexiglass beam
bar covered on scotch tape to avoid limb slip (1m length – 2.5cm width- 0.5m
elevation). At the beginning of the beam we place a little lamp and at the end
of the beam we place a dark box to motivate the animal to go towards the dark
side. 
2. Mice are allowed to explore the beam bar and receive two assisted trials to
cross it before the test. After two assisted trials, the test starts and animals
can traverse the entire length of the beam unassisted for two times. 
3. Measure the time the mouse takes to cross the beam. If the mouse has not
crossed the beam in 120 seconds, annotate 120 seconds for that animal. If the mouse
falls down, annotate as well 120 seconds. If the mouse does not cross or goes
backwards, remove them from the analysis. Calculate the average time after the
two trials to obtain the final results. 

4. Count the possible limb errors. An error is counted when, during a forward movement, a
limb (forelimb or hindlimb) slips through the beam. Classify them in right paw
errors or left paw errors. 
5. Clean the beam bar between animals.
TITLE: DNA extraction v9.0 (modified BOMB)
AUTHORS: Guan Jie Phang, Tsu-Chun Hung, Yin-Tse Huang

[DESCRIPTION]
DNA extraction using yttria-stabilized zirconia beads lysing and automated magnetic bead-based extraction.

[STEPS]
SECTION: Sample Collection
1. Add 200 µL of 0.5 mm beads to a 2mL screw tube.
  

SECTION: Sample Collection
2. Add 200 µL of 1 mm beads to a 2mL screw tube.
 
SECTION: Sample Collection
3. Add870 µL Lysis master mix to 2mL screw tube. The final look:
 
 

SECTION: Sample Collection
4. Collect 20-50 mg of sample to 2mL screw tube

 

SECTION: Sample lysis
5. Put the 2mL screw tube on vortex for sample lysis, at 3200 rpm  4 min 

 

SECTION: Centrifugation
6. Put 2mL screw tube in centrifuge for centrifugation, at this condition:10 x g, 3 min, 25 °C 
SECTION: DNA extraction
7. Add 350 µL of isopropanol to the 1st well of 96 well plate

   

SECTION: DNA purification
0.21. Add 100 µL of magnetic beads (10 mg/ml) to the 1st well of 96 deep well plate 
  
 


SECTION: DNA extraction
14. Add 300-500 µL of the sample (lysate) from the 1.5mL centrifuged tube to the 1st well of 96 deep well plate
  
 


SECTION: DNA extraction
9. Add 400 µL of isopropanol to the 2nd well of 96 deep well plate
  

SECTION: DNA extraction
10. Add 300 µL of 80% ethanol to the 3rd well of 96 deep well plate
 
SECTION: DNA extraction
11. Add 300 µL of 95% ethanol to the 4th well of 96 deep well plate
    

SECTION: DNA extraction
13. Add 100 µL of DEPC-treated water to the 6th well of 96 deep well plate 
 

SECTION: DNA extraction
15. Put the prepared 96-deep well plate in the automated DNA extraction machine (ZiXpress 32) and set up the settings as below.

SECTION: gDNA collection
17. After the extraction is done, put on the 96 magnetic plate to pellet the magnetic bead residues.
  

SECTION: DNA extraction
12. Add 300 µL of DDW to the 5th well of 96 deep well plate
  

SECTION: gDNA collection
18. Collect 100 µL of the eluted sample (avoid getting magnetic bead) as the DNA template for downstream experiments
  

SECTION: DNA extraction
8. Add 50 µL of magnetic beads (10mg/ml) to the 1st well of 96 deep well plate
  

SECTION: Program settings
16. The automated extraction program settings for ZiXpress 32.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Protocol to fix marine samples for flow cytometry analysis of phtoplankton and bacteria with SYBR-Green. <br />Fix at least 2 samples per depth sampled and at least 6 to 10 depths per vertical profile<br /><br /><strong>Reference<br /></strong><a href="https://www.researchgate.net/publication/265017015_An_Improved_Protocol_for_Flow_Cytometry_Analysis_of_Phytoplankton_Cultures_and_Natural_Samples" target="_blank">Marie, D., Rigaut-Jalabert, F. &amp; Vaulot, D. (2014). An improved protocol for flow cytometry analysis of phytoplankton cultures and natural samples. <em>Cytometry</em>. 85. p.pp. 962&ndash;968.</a>

[BEFORE_START]
<strong>Necessary equipment</strong><br /> - Vortex mixer <br />- Cryotubes 2 mL<br />- Liquid nitrogen tank

[STEPS]
?. 
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?. 
?. 
?. 
?. 
?.
TITLE: Lemna minor (duckweed) sterilization protocol
AUTHORS: Jason Laurich

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is designed to sterilize fronds of the common duckweed (</span><span style = "font-style:italic;">Lemna minor</span><span>) but should also be applicable to other members of the </span><span style = "font-style:italic;">Lemnoideae</span><span>. It involves both the surface sterilization of plants in bleach and testing for the presence of bacteria on plants through culturing in enriched media as a test for successful sterilization. </span></div></div>

[STEPS]
?. [Duckweed Sterilization : Preparation]
In a tea-strainer, strain duckweed plants under running water (doesn’t have to be sterile). This step gets rid of some surface algae.
?. [Duckweed Sterilization : Preparation]
Move surface-rinsed duckweed into a water bath; remove up to 50 plants from water bath and place in 50 mL Falcon tube approximately half full with autoclaved Phosphate Buffered Saline (PBS).
?. [Duckweed Sterilization : Preparation]
Vortex or shake intensely for 5 minutes.
?. [Duckweed Sterilization : Sterilization]
All of the subsequent sterilization steps should take place in a safety cabinet.
?. [Duckweed Sterilization : Sterilization]
Strain plants into a tea-strainer reserved for bleaching. Plants stuck to the side of the Falcon tube can be dislodge with distilled water.
?. [Duckweed Sterilization : Sterilization]
Place in (1% sodium hypochlorite) bleach for 60 seconds.
?. [Duckweed Sterilization : Sterilization]
Remove from bleach; rinse tea-strainer thoroughly by moving around in jar of autoclaved distilled water.
?. [Duckweed Sterilization : Sterilization]
Soak in autoclaved distilled water for 5 minutes or more. Repeat this step 3 times, in a new jar of fresh autoclaved distilled water each time.
?. [Duckweed Sterilization : Sterilization]
Open tea-strainer; pour autoclaved medium (e.g. Hoagland's, Krajncic medium) over tea-strainer to dislodge plants. This should be done over a clean, autoclaved jar (I use 500 mL Mason jars). Seal with lid and move to environmental chamber.
?. [Checking Duckweed Sterility]
Wait at least 3 days to give plants a chance to recover from the bleaching.
?. [Checking Duckweed Sterility]
In the safety cabinet (or other sterile location), transfer plants to autoclaved Mason jar approximately half full of your ENRICHED media of choice. I add 1 g yeast extract and 10 g sucrose per L to make it enriched.
?. [Checking Duckweed Sterility]
In as little as 2-3 days, you will know if your duckweed remains contaminated with bacteria — in this case, the media will become opaque as microorganisms replicate. If the media remains clear, congratulations!!! You have successfully sterilized your duckweed plants.
?. [Checking Duckweed Sterility]
If the media is cloudy, wash plants and transfer to Mason jar with autoclaved, unenriched media (in safety cabinet). After plants have recovered (ie. look green and healthy, after a week or more) you can re-sterilize them until all bacteria are removed.
TITLE: 10 Ways Tamra Judge CBD Gummies Can Improve Health! Reduce Chronic Pain!
AUTHORS: Tamra Judge CBD Gummies

[DESCRIPTION]
Tamra Judge CBD Gummies is a chewable gummy supplement that offers a variety of natural health benefits for stress relief. ORDER NOW THIS IS MY OFFICIAL WEBSITE!
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[STEPS]
TITLE: Strategies to Minimize Variability Between Individual qPCR Reactions
AUTHORS: Jonathan Phillips, David Chen, Gregor Blaha

[DESCRIPTION]
Precise pipetting is paramount for generating precise and reliable qPCR results. Despite meticulous pipetting, significant variability can arise in qPCR, even between technical replicates. While some variability is expected in samples with low target concentrations, it is concerning in samples with high target concentrations. To maintain data integrity, it is generally recommended to discard data with a discrepancy of more than 0.2-0.5 Ct values between technical replicates, in particular when the Ct values are below 30 [Anonymous, 2019]. 
This protocol provides approaches to reduce this variability through an optimized pipetting strategy. Central to this strategy is the use of pipetting schemes and built-in redundancies in the workflow keeping track of individual pipetting steps.

[STEPS]
TITLE: Data for manuscript: Selection of forage oat genotypes through GGE Biplot and BLUP
AUTHORS: Franklin Santos, Felix Marza

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>In Bolivia, there is a low predominance of forage oat productivity.  Therefore, it was proposed to select more productive and stable  genotypes through statistical methods of GGE Biplot and BLUP. The  research was conducted in three environments in Bolivia and six  commercial varieties of forage oats were evaluated; three of them  correspond to INIA Peru and the rest of Bolivia. Data were analyzed  through GGE Biplot and BLUP (Best Linear Unbiased Prediction) and an  average yield of 10.29 ±3.51 t ha</span><span style = "vertical-align:super;">−1</span><span> of dry matter was  obtained. BLUP accumulated greater variance than GGE Biplot in the first  two components. In terms of productivity and stability values, both  models have the same selection trend. Thus, Tayco and Texas were  selected for their outstanding characteristic in dry matter yield and  phenotypic stability.</span></div></div>

[STEPS]
TITLE: Taxon Group Protists: Barcoding 
AUTHORS: Estelle Kilias

[DESCRIPTION]
This protocol describes the DNA barcoding process for protists in the Darwin Tree of Life project.


[STEPS]
SECTION: DNA extraction
1. Extract genomic DNA from protists cultures using e.g. the DNeasy Plant or MagAttract kit (Qiagen) or equivalent and follow the manufacturer’s protocol/instructions. Depending on the taxa, DNA extraction can require an alternative pre-protocol approach, e.g. including a Plant DNAzol Reagent (Gibco BRL Life Technologies) or bead-beating step (10 ms for 3 min at 1 min intervals with 5 min rest on ice to avoid overheating).

DNeasy (Qiagen; 69204)
MagAttract (Qiagen; 67563)
SECTION: Barcoding
2. Amplify appropriate marker gene(s) (e.g. V4 or V9 region of 18S rRNA gene) using primer pairs as
appropriate for group/taxa using a Taq Master Mix (Qiagen), GoTaq polymerase (Promega), or equivalent with a final volume of 50 µL (see example below). For each marker region two primer combinations are available. For difficult templates, it might help to use high fidelity DNA Polymerase (e.g. Deep Vent DNA polymerase, New England BioLabs) to the mix.

Taq Master Mix (Qiagen; 201445)
GoTaq polymerase (Promega; M5001)
Deep Vent DNA polymerase (New England BioLabs; M0258S)

  

SECTION: Barcoding
2.1. 25-30 cyclesSpin down the tubes and run the PCR using an appropriately programmed thermal cycler.  
    

SECTION: Gel electrophoresis
3. Check the PCR products by gel electrophoresis against positive and negative controls and a 1kb
ladder. Load 4 µl PCR product with 1 µl loading dye on a 1% (w/v) agarose gel (SYBRSafe). Run
the gel at 110 mV for ~40 minutes.

SYBRSafe (Invitrogen; S33102)
SECTION: PCR purification
4. If one product is visible purify the PCR product using the QIAquick PCR purificastion kit or equivalent commercial PCR purification kits. If multiple products are visible on the gel, purify the PCR product using the QiaQick gel extraction kit (Qiagen) or equivalent commercial gel purification kits  If no products are visible, repeat PCR using the second primer pair for the marker gene region and follow the steps.

QIAquick PCR purification kit (Qiagen; 28104)
QiaQick gel extraction kit (Qiagen; 28704)
SECTION: DNA quantification
5. Determine the DNA concentration using a Qubit dsDNA HS Assay Kit (or equivalent). Ensure the
sample is 10-20 ng µl -1 for submission for sequencing.

Qubit dsDNA HS Assay Kit (Invitrogen; Q32851)
SECTION: Sanger sequencing
6. Submit for Sanger sequencing, do not clone the products prior to sequencing, sequence directly and inspect the chromatogram for evidence of dual amplicon sampling that would be indicative of contamination. For submitting to Eurofins (TubeSeq), mix 15ul of your purified PCR product (minimum concentration of 5ng/ul) and add 2ul of your primer (10uM). Ensure that the total volume is not more or less than 17 ul.