eamag/protocols-with-steps huggingface dataset:
TITLE: Production of Crude AAV Virus Extract
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol is used to produce crude preps of AAV of any serotype.

Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Numbers U19MH114830, RF1MH114126, and RF1MH121274. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

[STEPS]
TITLE: Fecal Water Content Assay
AUTHORS: Sabina Marciano, Roberta Marongiu

[DESCRIPTION]
This test is used to check for signs of constipation


[STEPS]
1. Label, weight and record the weight of each Eppendorf tube
2. Collect 2 pellets from each mouse and weigh. 
3. Dry the pellets in the oven at 60 degrees C for 24 hrs
4. Weigh the pellets again after drying
5. Calculate fecal water content according to the equation: 

(fecal weight before drying − fecal weight after drying)/feces weight before drying × 100.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>We have developed a multiplexed single molecule FISH protocol for use at the Institute. This protocol was optimized on human tissue, but will work on mouse tissue as well. It was adapted from Lyubimova et. al., Nature Protocols, 2013. </p>

[BEFORE_START]
<div>
<div>
<div>
<div>
<div>
<p>Ensure all incubators and ovens are at the appropriate temperature prior to experiment. </p>
</div>
</div>
</div>
</div>
</div>
<div> </div>

[GUIDELINES]
<p>Ensure that all reagents are in recombinant and RNAse-free format, as we have noticed RNA degradation in solutions that contain enzymes derived from whole organisms. </p>
<p> </p>
<p>We filter every solution with a 0.2um syringe filter prior to use. This reduces background spots and dust that interfere with imaging of diffraction limited spots. </p>
<p> </p>
<p>For the SDS treatment after fixation and permeabilization, be gentle when dropping SDS onto the section, as well as during washes. This treatment is relatively harsh and the tissue must be treated somewhat delicately. </p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol provides the detailed methods of assembly and annotation of the R. crenulata genome.</p>

[BEFORE_START]
<p>Get raw sequencing data in Fastq format.</p>

[STEPS]
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TITLE: Confidentiality and disclaimer examples
AUTHORS: Lenny Teytelman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Yale's SalivaDirect protocol</span><span>: "“For diagnostic and screening purposes, only labs designated by Yale and that follow the Instructions For Use, which we will provide, can use SalivaDirect. For research purposes, our open-source protocol can be used.”</span></div><div class = "text-block"><span style = "font-weight:bold;">Suggestion: </span><span>“protocols in this collection have not been tested/verified by the staff at the CDC.”</span></div><div class = "text-block"><span style = "font-weight:bold;">Suggestion: "Do not disseminate. </span><span>Only labs designated by TOAST should use these instructions."</span></div></div>

[STEPS]
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol provides a method for assembly of metagenomic data using the Ray assembly toolkit and the subsequent analysis of contig statistics. Based on the publication: <br /><br />Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." <em>mBio</em> 6.5 (2015): e01578-15.

[BEFORE_START]
Perl scripts and other supplementary information available at: <br /><br /><a href="https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248" target="_blank">https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248</a>

[GUIDELINES]
<strong>Required Software:</strong><br />
<ul>
<li>Ray-2.3.1</li>
<li>bowtie2-2.1.0</li>
</ul>
<strong>Relevant Files</strong><br />Output:<br />
<ul>
<li>Virome_Sequence_Counts</li>
<li>Whole_Microbiome_Sequence_Counts</li>
</ul>
Perl Scripts: calculate_abundance_from_sam.pl<br />R Scripts: <a href="https://www.protocols.io/view/Script-R1-Virome-Contig-and-Sequencing-Statistics-eh5bb86?step=undefined" target="_blank">R1</a> and <a href="https://www.protocols.io/view/Script-R2-Whole-Metagenome-Contig-and-Sequencing-S-eh6bb9e" target="_blank">R2</a><br />Python Scripts: get_trimmed_pairs.py

[STEPS]
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TITLE: Iodine staining of starch to determine amylose percentage
AUTHORS: Chris Berndsen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Starch is well known to complex with iodine producing strong absorbance between 500 and 750 nm. Two structures that compose structure, amylose and amylopectin complex with iodine differently resulting in distinct absorption spectra. The staining is blue for amylose, brown for amylopectin, and yellow for soluble polymers of glucose (ex. glycogen).</div><div class = "text-block">Measuring iodine-stained samples at 525 nm and 700 nm allows for the calculation of the percent amylose. </div></div>

[STEPS]
?. [Preparing starch for spectroscopic measurement]
Obtain soluble starch or starch granule sample in sterile water
?. [Preparing starch for spectroscopic measurement]
Warm sample to  in a water bath for
95 °C
Starch granules will gelatinize under these conditions. Do not cool the samples otherwise staining may not work efficiently
?. [Preparing starch for spectroscopic measurement]
Add  of starch solution to a cuvette.
20 µl
Plastic cuvettes will become stained with iodine solution and cannot be reused.
?. [Preparing starch for spectroscopic measurement]
Add  of Iodine solution. Mix by inverting.
980 µl
?. [Measuring absorbance of iodine stained samples]
Measure absorbance at 525 nm and 700 nm.  Record values in the table. Add more columns for each new sample and edit the column name to indicate the sample.WavelengthAbsorbance525 nm700 nm
WavelengthAbsorbance525 nm700 nm
?. [Measuring absorbance of iodine stained samples]
Add  of water or whatever buffer the starch is dissolved in to a new cuvette and then add  of the iodine solution. Mix by inverting.
20 µl
980 µl
?. [Measuring absorbance of iodine stained samples]
Measure absorbance at 525 nm and 700 nm.WavelengthAbsorbance525 nm700 nm
WavelengthAbsorbance525 nm700 nm
?. [Calculating percentage amylose]
Subtract the absorbance of water-iodine measured in Step 7 from the absorbance for the sample(s) containing starch.
?. [Calculating percentage amylose]
The percentage of the sample that is amylose is determined from the equation below:percent ~amylose = \frac{3.039 - (7.154 \times \frac{OD_{700}}{OD_{525}})}{(3.048 \times \frac{OD_{700}}{OD_{525}}) - 19.192}
?. [Calculating percentage amylose]
Record your calculations in the table below.Sample name% amylose
Sample name% amylose
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>&nbsp;Primers: </strong><br />GM3:&nbsp;&nbsp;&nbsp;&nbsp; AGA GTT TGA TCM TGG C &nbsp;These primers are for almost full-length 16s (8F/1492R) <br />GM4:&nbsp;&nbsp;&nbsp;&nbsp; TAC CTT GTT ACG ACT T &nbsp; &nbsp; <br /><br />
<table style="width: 250px; height: 250px;">
<tbody>
<tr>
<td colspan="2"><strong>Reaction mix for GM3/GM4</strong></td>
</tr>
<tr>
<td>Water</td>
<td>10.50 &mu;l</td>
</tr>
<tr>
<td>Primer GM3, 10&nbsp;&mu;M</td>
<td>0.50 &mu;l</td>
</tr>
<tr>
<td>Primer GM4, 10&nbsp;&mu;M</td>
<td>0.50 &mu;l</td>
</tr>
<tr>
<td>2x Go Taq Green</td>
<td>12.50 &mu;l</td>
</tr>
<tr>
<td>DNA</td>
<td>1.00 &mu;l</td>
</tr>
<tr>
<td>&nbsp;</td>
<td>Total=25 &mu;l</td>
</tr>
</tbody>
</table>
<br />
<table style="width: 250px; height: 250px;">
<tbody>
<tr>
<td colspan="3"><strong>Cycling with GM3/GM4</strong></td>
</tr>
<tr>
<td>95 &deg;C</td>
<td>5 min</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>95 &deg;C</td>
<td>15 sec</td>
<td rowspan="3">30 cycles</td>
</tr>
<tr>
<td>48 &deg;C</td>
<td>15 sec</td>
</tr>
<tr>
<td>72 &deg;C</td>
<td>2 min</td>
</tr>
<tr>
<td>72 &deg;C</td>
<td>5 min</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>15 &deg;C</td>
<td>Hold</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>

[STEPS]
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>Primers:<br /><br /></strong>381_Lysis_418_F:     TGC CTG AAC GGC TTA GAG GTT TGC<strong><br /></strong>381_Lysis_418_R:     AGC ACC AAT CGG CGT CTT CGT <br /><br />Putative Lysis gene product = 350 bp<br /><br /><strong><strong>Reaction Mix<br /><br /></strong></strong>
<table>
<tbody>
<tr>
<td>Water</td>
<td>10.5-6.5μl</td>
</tr>
<tr>
<td>GoTaq Green</td>
<td>12.5μl</td>
</tr>
<tr>
<td>Primer F, 10μM</td>
<td>0.5μl</td>
</tr>
<tr>
<td>Primer R, 10μM</td>
<td>0.5μl</td>
</tr>
<tr>
<td>DNA template</td>
<td>1.0-5.0μl</td>
</tr>
<tr>
<td>Total</td>
<td>25μl</td>
</tr>
</tbody>
</table>
<br /><strong>Cycling</strong><br /><br />
<table>
<tbody>
<tr>
<td>95°C</td>
<td>5 min</td>
<td>(try 98°C for sorted samples to bust open capsids)</td>
</tr>
<tr>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>95°C</td>
<td>30 sec</td>
<td> </td>
</tr>
<tr>
<td>59.9°C</td>
<td>30 sec</td>
<td>30 cycles</td>
</tr>
<tr>
<td>72°C</td>
<td>90sec</td>
<td> </td>
</tr>
<tr>
<td> </td>
<td> </td>
<td> </td>
</tr>
<tr>
<td>72°C</td>
<td>5-10min</td>
<td> </td>
</tr>
<tr>
<td>15°C</td>
<td>Hold</td>
<td> </td>
</tr>
</tbody>
</table>
<strong><br /><br /></strong>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Dimethoxypropane (DMP) is an organic compound that reacts with water under acidified conditions to form methanol and acetone. The compound can also be used in place of an ethanol series to dehydrate formalin-fixed plant tissues for serial sectioning.</p>

[BEFORE_START]
<p>Prepare and fix the tissue according to your normal lab protocol. I have been using fixation in FAA under a vacuum followed by a wash with 50% ethanol, but fixation in ethanol:acetic acid also works. I have done all of these steps in 25mL glass scintiallation vials. Additionally, to keep track of samples through all the solution changes, we place a small paper label (written in pencil) in the vial with the samples.</p>

[STEPS]
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TITLE: Adhesive Removal Test to assess sensorimotor deficits in parksisonian mice
AUTHORS: natalia.lopezgonzalezdelrey, Elizabeth Phelan, Zachary Gaertner

[DESCRIPTION]
This behavior is used to asses fine motor movements in a mouse parkinsonian model. It checks for correct paw and mouth sensitivity (time-to-contact) and correct dexterity (time-to-remove).

[STEPS]
SECTION: Adhesive Removal Test
1. 
Total duration: 4 days
SECTION: Protocol
2. Place the animal cage in the testing room to allow the animals to acclimate. 60 min

SECTION: Protocol
3. Acclimating mice to testing box. 1 min 

SECTION: Protocol
4. Apply the two adhesive tape strips with equal pressure on each animal paw so that they cover the hairless part of the forepaws. Place the adhesive in alternate order (right or left) each day and randomize it within each group. 3 min

SECTION: Protocol
5. Video record the animal’s behavior in the testing cage. 2 min

SECTION: Protocol
6. The contact time is defined as the point that the mouse reacts to the presence of the adhesive tape strips. For each adhesive, the mouse may start by either shaking its paws and/or directly bring its paws to its mouth. Both behaviours indicate that the mouse has felt the adhesive tape and represent the end of the time to contact. Time 1: when the mouse has felt the first piece of tape. Time 2: when the second piece of tape is felt.
SECTION: Protocol
7. Continue to observe the animal’s behavior in the testing cage. The mouse may use its mouth, and to a lesser extent the other forelimb, to attempt to remove the adhesives. Time 3: when the first piece of tape is removed. Time 4: when the second piece of tape is removed. Distinguish between right and left paw for comparisons.
SECTION: Analysis
9. Different analysis can be made:
- Mean Time to Notice (T1,T2)
- Mean Time to Remove (T3,T4 - T1,T2)

Compare

Tip: in the post-analysis: you can distinguish between right and left paw for comparisons. This helps to detect limb differences.
Example:
- Mean Time to Notice Right (T1)
- Mean Time to remove Right (T3)
- Mean Time to Notice Left (T2)
- Mean Time to remove Left (T4)


SECTION: Protocol
8. Return the mouse to its cage. After 24 h, repeat the procedure.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Background: To improve the care and treatment of HIV-exposed children, early infant diagnosis (EID) using dried blood spot (DBS) sampling has been performed in Senegal since 2007, making molecular diagnosis accessible for patients living in decentralized settings. This study aimed to determine the evolution of the HIV transmission rate in children from 2008 to 2015 and to analyze associated factors, particularly the mother’s treatment status and/or child’s prophylaxis status and the feeding mode. 
Methods: The data were analyzed using EID reports from the reference laboratory. Information related to sociodemographic characteristics, HIV profiles, the mother’s treatment status, the child’s prophylaxis status, and the feeding mode was included. Descriptive statistics were calculated, and bivariate and multivariate logistic regression analyses were performed. 
Results: During the study period, a total of 5418 samples (5020 DBS and 398 buffy coat) from 168 primary prevention of HIV mother-to-child transmission (PMTCT) intervention sites in Senegal were tested. The samples were collected from 4443 children with a median age of 8 weeks (1-140 weeks) and a sex ratio (M/F) of 1.1 (2309/2095). One-third (35.2%; N = 1564) of the children were tested before 6 weeks of age. Twenty percent (N = 885) underwent molecular diagnostic testing more than once. An increased number of mothers receiving treatment (57.4%; N = 2550) and children receiving prophylaxis (52.1%; N = 2315) for protection against HIV infection during breastfeeding was found over the study period. The transmission rate decreased from 14.8% (95% confidence interval (CI): 11.6-18.7) in 2008 to 4.1% (95% CI: 2.7-6.2) in 2015 (p < 0.001).
Conclusion: This study demonstrates the effectiveness of PMTCTinterventions in Senegal and indicates that increased effortsshould be continued to reduce the MTCT rate to less than 2%. 


[STEPS]
?.
TITLE: No Title
AUTHORS: 

[BEFORE_START]
You must determine the titer of your phage lysate before performing the experiment.&nbsp;

[GUIDELINES]
Note: one-step growth experiment instructions are for MOI=3.

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This collection contains the protocols and recipes for the preparation of cell-free RNAPT7 transcription/translation coupled reactions.</p>
<p>The crude extracts are obtained from IPTG-treated E.coli BL21 DE3 STAR cells. IPTG triggers RNAPT7 expression and accumulation in cells before collection. Reactions take place when the cell extract is mixed with a maltodextrin-based energy solution along with amino acids solution and other salts (Magnesium and Potassium). This system allows transcription/translation coupled reactions from plasmid DNA that contains T7 promoter transcriptional units. In our hands, it has been possible to express different fluorescent reporters, including deGFP which is highly efficient. Products of the reaction can be measured during the whole reaction time in a plate reader with appropriate optic filters, open hardware cameras, and microscopes. When set up correctly, deGFP fluorescence intensity is detected as fast as ∼30 minutes after the reaction starts and it shows a rapid increase during the first ∼2.5-3.0 hours. </p>
<p> </p>
<p>The system presented here contains the features highlighted bellow, and is an adaptation of protocols and findings from the following listed papers:<br /><br /><strong>1.- This system uses maltodextrin and Polyphosphate-based energy solution: These reagents do not need refrigeration while transporting.</strong></p>
<p><br />Kim, H.-C., Kim, T.-W., Kim, D.-M., . Prolonged production of proteins in a cellfree protein synthesis system using polymeric carbohydrates (2011)<br /> <br />Caschera, F.; Noireaux, V., A cost-effective polyphosphate-based metabolism fuels an all E. coli cell-free expression system. Metabolic engineering (2015)<br /> <br /><strong>2.- The S12 crude extract is prepared from E.coli cells ( expressing Pol T7 ) using bead beater. We use an S12 extract which is simple and d not require ultra-centrifugation nor dialysis</strong><br /> <br />Sun, Z. Z.; Hayes, C. A.; Shin, J.; Caschera, F.; Murray, R. M.; Noireaux, V., Protocols for implementing an Escherichia coli based TX-TL cell-free expression system for synthetic biology. Journal of visualized experiments (2013)<br /> <br />Kim, TW., Kim, HC., Oh, IS. et al. A highly efficient and economical cell-free protein synthesis system using the S12 extract of Escherichia coli. Biotechnol Bioproc E (2008)<br /> <br /><strong>3.- The amino acid stock solution is highly concentrated</strong><strong> (each amino acid is at 12 nM in a stable mix)</strong><strong>:</strong><br /> <br /> <br />Caschera, V. Noireaux Preparation of amino acid mixtures for cell-free expression systems. Benchmarks (2015)</p>
<p> </p>

[STEPS]
TITLE: Protocol for Exonuclease VII (NEB #M0379)
AUTHORS: New England Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Exonuclease VII efficiently cleaves single-stranded DNA (ssDNA) from both 5´→3´ and 3´→5´ direction. This enzyme is not active on linear or circular dsDNA</div></div>

[STEPS]
?. Set-up the reaction as follows:  AB1COMPONENTSReaction Volumes2&nbsp;PCR Product5&nbsp;μl3&nbsp;Exonuclease VII&nbsp;2 μl (20 units)
AB1COMPONENTSReaction Volumes2&nbsp;PCR Product5&nbsp;μl3&nbsp;Exonuclease VII&nbsp;2 μl (20 units)
?. Incubate the mix at  for .
37 °C
?. Heat inactivate  for  prior to Sanger DNA sequencing. For other downstream molecular cloning applications column cleanup is recommended.
95 °C
Note: See Exo-CIP™ Rapid PCR Cleanup Kit (NEB #E1050) for Sanger Sequencing, SNP detection, or library preparation for NGS.
?. To clean up treated samples, we recommend using one of the following steps:a. Column clean up (we recommend the Monarch® PCR & DNA Cleanup Kit, NEB #T1030), orb. Running the reaction on an agarose gel, and then extracting the DNA (we recommend the Monarch Gel Extraction Kit, NEB #T1020), orc. Performing a phenol/chloroform extraction followed by ethanol precipitation.
TITLE: Washington University SenNet Case Collection Protocol
AUTHORS: Daniel Rapp, Matthew Wyczalkowski

[DESCRIPTION]
This document outlines inclusion and exclusion criteria for healthy participants for the WUSTL Senescence Network (SenNet) by the Tissue Mapping Center (TMC) at Washington University. 

[STEPS]
1. Study coordinator will receive surgeon schedules at the beginning of each week. All surgeons should be IRB approved for the study.
4. Study coordinator will evaluate patient for eligibility using criteria detailed in following sections. 
2. Study coordinator will call potential study participants over phone using IRB approved phone script to see if participants are interested. Other option is to approach potential participants in Clinic when they are meeting with surgeon before the surgery date. Discuss with surgeon team which is best option as to not disrupt clinic flow.
3. Once patient is identified and interested, go over IRB approved consent form with patient. Answer all questions and allow for time if they need to think the process over. Must give patient a signed copy of consent form for their own personal records. Enter subjects into Oncore within 24 hours of consent process.
SECTION: Inclusion Criteria
5. Able to understand and willing to sign an IRB-approved written informed consent document
Undergoing routine surgery under general anesthesia for non-cancer diagnosis, including: 
Laparoscopic upper gastrointestinal surgery (for benign diagnosis, e.g. cholecystectomy, hernia repair) or Open abdominal surgery for similar indications. (need access to liver and omental fat)
18 years of age or older
All races and ethnicities eligible
All BMIs eligible
Equal number of female and male donors will be accepted
Past history of cancer and chemo are eligible
SECTION: Exclusion Criteria
6. Undergoing a Cancer surgery
Currently receiving Chemo treatment
Positive for HIV, Hepatitis B or C virus
Under 18 years of age
Unable to fully understand/sign consent form. (e.g. language barrier, compromised mental status)
SECTION: Documents
7. Signed consent document saved and stored in secure location. Copy of signed consent also given to patient
Signed W9 tax form saved and uploaded for payment of participant.
Check request form signed by PI saved and uploaded for payment of participant.
TITLE: Enzo’s CGH Labeling Kit for Oligo Arrays
AUTHORS: Hendrik F van Essen

[STEPS]
?. [Denature DNA and anneal random primers]
pipette the following in a nuclease free thermocycler strip:a.	DNA	         X µlb.    water	19 – X µl	(Vial W) b.	Primers/Reaction buffer	20 µl	(Vial 1) Total volume:	39 µl
?. [Denature DNA and anneal random primers]
Add cap, flick to mix contents and briefly spin down
?. [Denature DNA and anneal random primers]
Heat at  for
?. [Denature DNA and anneal random primers]
Centrifuge briefly
?. [Denature DNA and anneal random primers]
Place in a cooled 96 well rack on ice  for
?. [Extend Primers with Klenow Exo-DNA Polymerase]
Add, while samples are on ice :a.	To test sample	        10 µl     Cy3	(Vial 2)b.	To reference sample	10 µl     Cy5	(Vial 3)c.	Klenow Exo-DNA Pol	  1 µl	        (Vial 4)                                Total	50 µl
?. [Extend Primers with Klenow Exo-DNA Polymerase]
Add cap, flick to mix contents and briefly spin down
?. [Extend Primers with Klenow Exo-DNA Polymerase]
Incubate at for
?. [Extend Primers with Klenow Exo-DNA Polymerase]
Add 5µl of Stop Buffer (Vial 5), mix, and briefly centrifuge
?. [Removal of uncoupled nucleotides]
Transfer labelled DNA’s to 1.5 ml reaction tubes and keep Cy3 and Cy5 labelled DNA’s separated
?. [Removal of uncoupled nucleotides]
Add 275 µl of Binding Buffer (PB)
?. [Removal of uncoupled nucleotides]
Load column and centrifuge for 60 seconds at 16,000g and discard flow through
?. [Removal of uncoupled nucleotides]
Add 650 µl of Wash Buffer (PE), centrifuge 60 seconds at 16,000g and discard flow through
?. [Removal of uncoupled nucleotides]
Centrifuge for 60 seconds at 16,000g, discard centrifuge tube and place column in fresh 1.5 ml centrifuge tube
?. [Removal of uncoupled nucleotides]
Add 10.5 µl of Elution Buffer (EB) (middle of the filter) and incubate for 1 minute.
?. [Removal of uncoupled nucleotides]
Centrifuge for 60 seconds at 16,000g
?. [Removal of uncoupled nucleotides]
Measure yield and specific dye incorporation with NanoDrop, Program MicroArray. Labelled, cleaned up gDNA can be stored at -20°C in the dark for 7 days.
?. [Removal of uncoupled nucleotides]
Add 10.5 µl of Elution Buffer (EB) (middle of the filter) and incubate for 1 minute. .