eamag/protocols-with-steps huggingface dataset:
TITLE: Quick Protocol for DNA Cleanup and Concentration Using the Monarch® PCR &amp; DNA Cleanup Kit (5 μg) (NEB #T1030)
AUTHORS: New England Biolabs

[DESCRIPTION]
This is the "quick" version of Monarch® PCR & DNA Cleanup Kit (5 μg) Protocol (NEB #T1030) DNA Cleanup and Concentration. For the full protocol, please click here.

[BEFORE_START]
Add isopropanol to Monarch DNA Cleanup Binding Buffer prior to use*:

For the 50-prep kit, add 14 mL of isopropanol to the DNA Cleanup Binding Buffer.
For the 250-prep kit, add 63.5 mL of isopropanol to the DNA Cleanup Binding Buffer.

Add ethanol to Monarch DNA Wash Buffer prior to use (4 volumes of ≥ 95% ethanol per volume of Monarch DNA Wash Buffer)

For the 50-prep kit, add 20 mL of ethanol to the Monarch DNA Wash Buffer
For the 250-prep kit, add 100 mL of ethanol to the Monarch DNA Wash Buffer

Always keep all buffer bottles tightly closed when not in use.

All centrifugation steps should be carried out at 16.000 x g (~13.000 rpm).



[GUIDELINES]
For detailed protocol and more information, visit www.neb.com/T1030

The full protocol is available here.

The video protocol is available here.

 
There are two protocols available for this product:

DNA Cleanup and Concentration (below): for the purification of up to 5 µg of DNA (ssDNA > 200 nt and dsDNA > 50 bp) from PCR and other enzymatic reactions.

Oligonucleotide Cleanup: for the purification of up to 5 µg of DNA fragments ≥ 15 bp (dsDNA) or ≥ 18 nt (ssDNA). Expected recovery is > 70%. When purifying ssDNA of any size, recovery can be increased by using this protocol; however, it is important to note that this protocol shifts the cutoff for smaller fragments to 18 nt (rather than 50 nt for the DNA Cleanup and Concentration Protocol). A detailed protocol and quick protocol are available for your convenience.
A step-by-step protocol is available here.


[STEPS]
1. Dilute sample with DNA Cleanup Binding Buffer according to the table below. Mix well by pipetting up and down or flicking the tube. Do not vortex.  
   Sample Type Ratio of Binding Buffer: Sample Example   dsDNA > 2 kb (plasmids, gDNA) 2:1 200 μl: 100 μl   dsDNA < 2 kb (some amplicons,  fragments) 5:1 500 μl: 100 μl   ssDNA > 200 nt* 7:1 700 μl: 100 μl   

 
2. Insert column into collection tube and load sample onto column. Spin at 16000 x g for 1 min, then discard flow-through.
3. Re-insert column into collection tube. Add 200 µL and spin at 16000 x g for 1 min.
 
4. Repeat Step 3: Re-insert column into collection tube.  Add 200 µL and spin at 16000 x g for 1 min.
 

5. Transfer column to a clean 1.5 ml microfuge tube. 
 
6. Add ≥ 6 µL to the center of the matrix. Wait for 1 min.
7. Spin for 1 min to elute DNA.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>PFGE Protocol is from Griffith 2000</strong><br /><br /><strong>PFGE Modifications (Schwalbach 2002):&nbsp;</strong><br />- 1% Seakem Gold agarose gels are used. Gel is poured after it has cooled to below 45&deg;C. <br />- Instead of using 1:10 TE for the 3 microcon rinses, regular TE (pH 7.6) is used. <br />- For the final collection of viruses from microcons use two 240 x g spins with 10 &mu;l of TE each spin instead of one 5 minute 1000 x g spin with 20 &mu;l 0.5x TBE.&nbsp;<br />- NEB PFGE standards are occasionally used, though liquid lambda/5 kb ladders are also used. <br />- Gels are destained in diH<sub>2</sub>O for minimum of 30 minutes.&nbsp;<br />- Should use the majority of the 150 and 500 m samples. (i.e. all of it)

[STEPS]
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TITLE: HA Filtration of Wastewater for SARS-CoV-2 detection
AUTHORS: Vbarua , Md Ariful Islam Juel, Neha Mittal, Nick Stark, Cynthia Gibas, jschluet , Mariya Munir

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol provides the information on how to concentrate SARS-CoV-2 virus in wastewater sample using a vacuum filtration in conjunction with electronegative filter (type HA) method. The volume of wastewater sample that could be filtered at once depends on the turbidity of the sample. </div></div>

[STEPS]
?. Put on proper PPE (lab coat, N95 fitted masks, gloves, face shield) before handling the wastewater sample.
?. The biosafety cabinet should be properly sterilized (UV light for 20 minutes and the surface should be cleaned with 70% ethanol). Gloves should be sprayed with 70% ethanol prior to starting.
?. Place a 40 mL aliquot of wastewater into a 50 mL conical tube.Add 4 µL of Bovine Coronavirus modified live vaccine (BCoV)  to 40 mL of wastewater.
?. Adjust the pH of the wastewater sample to 3.5-4 using 10 M HCl either with pH paper or pH meter. While adding the HCl the sample should be properly mixed.N.B. If there are multiple samples pH paper is more favorable to avoid cross-contamination by using the same pH electrode for every sample. If pH meter is used electrode should be cleaned with 10% bleach, followed by 2% sodium thiosulfate and distilled water.
?. Add 400 µL of 2.5M MgCl2 x 6 H2O per 40 mL of sample, mix the sample properly.
?. Pass the entire volume of sample through a disposable filter funnel fitted with electronegative (type HA) filter of 0.45 μm pore size and 90 mm diameter with a vacuum filtration manifold .
?. Following filtration, fold the filter paper using sterilized forceps dipped in 70% ethanol and place in the labeled Eppendorf tube containing 1 mL of lysis buffer (AVL with carrier RNA, Qiagen).
?. Repeat steps 1- 7 for each replicate. N.B. For the same sample, the filter funnel can be the same, but the filter paper should be new for every replicate. But for different sample the filter funnel and the filter paper should be changed every time.
?. For each set of wastewater samples collected and processed, a method blank was prepared.N.B. Add 4 µL BCoV to 40 mL phosphate buffer (1X PBS) and repeat step 5-7.
?. After completing the filtration work, clean the biosafety cabinet with 10% bleach and 70% ethanol and turn on the UV lights for 20 minutes.
?. Add 10% bleach to any liquid waste generated during the process and let it sit for 30 minutes before disposing.
TITLE: Western Blot
AUTHORS: Yiqin Shen

[DESCRIPTION]
This is a protocol of Leo Parra-Rivas, PhD.

[STEPS]
SECTION: Preparing Proteins
1. Dilute protein samples to 1.25 µg/µL. For running a 10-well gel, each loading sample should be around 15 µLto 20 µL.

SECTION: Transferring the Gel
5. Assemble the module according to instructions (membrane = 0.2ul pores; gel needs to be flipped). Ensure all bubbles are removed. 
Add transfer buffer (ice cold) to the tank. Securely locate the module. Add ice around the tank before running. 
Run at 25-30V for 85 min

SECTION: Staining
6. Fixation: rinse membrane with 1xPBS. Incubate in 0.4% PFA for 30 min.

SECTION: Preparing Proteins
2. Add 4x sample buffer to protein samples. 
SECTION: Preparing Proteins
3. Denature protein at 95-100 °C for 5 min to10 min 

SECTION: Running the Gel
4. Secure the gel (NuPAGE 4-12% Bis-Tris Gel; 1.5mm 10 wells) into the module. Fill the tank with running buffer. Load 15 µL of the ladder, and 20 µLof the samples (25ug/20ul, more if less concentrated). 
Run at 70-80V for 75 min. Until the blue band reaches the end of the gel, run for 10 more min. 

SECTION: Running the Gel
4.1. Ensure the strip on the bottom is removed. Remove the comb without breaking the wells.
SECTION: Running the Gel
4.2. Running Buffer: 1900ml H2O, 100ml Running buffer 20X
SECTION: Running the Gel
4.3. You may add more buffer if the buffer level is low. 
You may increase the voltage to 100V. 
SECTION: Staining
7. Blocking: wash with 1xPBS, 3 times 10 min. Block with 5-10% milk for 60 min. 

SECTION: Staining
8. Primary antibody: Specific primary antibody diluted in 5% nonfat dried milk in TBST, 4 °C overnight with rocker. 
Wash: Recover the primary antibody, and rinse with wash buffer. Wash for 30 min with wash buffer

SECTION: Staining
9. Secondary antibody: Secondary antibody 1:2000 in milk, 60 min Room temperature. Add approximately 10 ml to each tray.
Wash: Rinse and wash with wash buffer. The membrane can be stored in the wash buffer
SECTION: Imaging
10. Mix imaging reagent 1:1. Either apply reagent to membrane before imaging, or incubate the membrane in mixed reagent for 5 min while rocking.
Avoid bubbles when placing the membrane on the machine. 
Single image, start with 0.1s exposure and gradually increase until desired exposure is achieved.
More reagent can be added if signal is reduced.

SECTION: Transferring the Gel
5.1. transfer buffer: 1500ml H2O, 400ml methanol, 100ml transfer buffer 20X
SECTION: Staining
8.1. Wash buffer: 1800ml H2O, 200ml 10X PBS, 2ml Tween-20
TITLE: Dry cell weight by centrifugation
AUTHORS: Joao Vitor Molino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocols describe the steps required for dry cell weight measurements using microcentrifugal tubes. </div></div>

[STEPS]
?. [Material]
Analytical balance with high precision (The higher the precision the better. For example a balance with a 0.1mg readability, could account to approximately 10% error alone in a measurement of 1mL sample of a culture at 1g/L) Microcentrifugal tubesMicrocentrifuge
?. [Tubes preparation]
Label microcentrifugal tubes Dry the tubes at   ,   Cool tubes at   for   Record the weight of the tubesDry the tubes at  , Cool tubes at  for
90 °C
90 Room temperature
?. [Weighting the dry cell material]
Harvest 2 mL of culture in a previously weighted tubeCentrifuge the sample at   Carefully remove the supernatant by pipettingWash the cells with ddH20, and centrifuge the sample at   Carefully remove the supernatant by pipettingDry the tubes at   ,   Cool tubes at   for   Record the weight of the tubesSubtract the initial tube weight to achieve the dry cell weightCentrifuge the sample at Carefully remove the supernatant by pipettingWash the cells with ddH20, and centrifuge the sample at   Carefully remove the supernatant by pipettingDry the tubes at   ,   Cool tubes at   for   Record the weight of the tubesSubtract the initial tube weight to achieve the dry cell weightWash the cells with ddH20, and centrifuge the sample at Carefully remove the supernatant by pipettingDry the tubes at   ,   Cool tubes at   for   Record the weight of the tubesSubtract the initial tube weight to achieve the dry cell weightDry the tubes at  , Cool tubes at  for Record the weight of the tubesSubtract the initial tube weight to achieve the dry cell weight
Centrifuge: 20000 1324, 1 min, 25 10
Centrifuge: 20000 1324, 1 min, 25 10
90 °C
90 Room temperature
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This procedure is used to dissociate adult (10 wk.) mouse pancreas into single cells. The procedure is carried out on ice in order to maintain a more authentic gene expression profile. It is a two-layered dissociation, with each layer consisting of 5 mg/mL type 4 collagenase. The yield is 4400 cells/mg with 94% viability. </p>

[BEFORE_START]
<div>
<div>
<div>
<div>
<div>
<p>-Prepare enzyme mixes and leave on ice. </p>
<p>-Cool centrifuges to 4 °C.</p>
<p>-Isolate and transport tissue in ice-cold DPBS. </p>
</div>
</div>
</div>
</div>
</div>
<div> </div>

[GUIDELINES]
<p><strong>Type 4 Collagenase Enzyme Mix (2 x 1 mL)</strong></p>
<p>50 µL Type 4 Collagenase 100 mg/mL (5 mg/mL final conc.)</p>
<p>5 µL DNAse (125 U)</p>
<p>5 µL 1 M CaCl<sub>2</sub> (5 mM final conc.)</p>
<p>10 µL 10% BSA/PBS (0.1% BSA final conc.)</p>
<p>930 µL DPBS</p>
<p> </p>
<p><strong>Reagents</strong></p>
<p>Collagenase Type 4 - Worthington (LS004186) - 100 µL aliquots of 100 mg/mL, frozen at -80 °C</p>
<p>DNAse 1 - Applichem (A3778) – 10 µL aliquots each with 250 U, frozen at -80 °C</p>
<p>DPBS - ThermoFisher (cat. #14190)</p>
<p>Red Blood Cell Lysis Buffer - Sigma (R7757)<br /> Trypan Blue Solution 0.4% - Gibco (15250061)<br /><br /></p>
<p><strong>Required Equipment &amp; Consumables:</strong></p>
<p>Refrigerated centrifuge</p>
<p>Pipettes and pipet tips (MLS)<br />15, 50 ml Conicals (MLS)<br />1.5 mL tubes (MLS)<br />30 µM filters (MACS SmartStrainers, 130-098-458)</p>
<p>Petri dishes (MLS)<br />Razor blades (MLS)<br />Ice bucket w/ice (MLS)<br />Hemocytometers - InCyto Neubauer Improved (DHC-NO1-5)</p>
<p> </p>
<p><strong>The protocol workflow is as follows: </strong></p>
<ol>
<li>Isolate pancreas</li>
<li>First layer</li>
<li>Second layer</li>
<li>Preparing cells for Chromium/DropSeq</li>
</ol>

[STEPS]
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TITLE: Empty protocol reagent
AUTHORS: Darja Darja

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

[STEPS]
?. Po podatkih uprave za zaščito in reševanje so nedeljska neurja prizadela  območja 46 občin, zaznali so več kot 100 dogodkov. Najhuje je bilo v občinah Zagorje, Rogatec in Rogaška Slatina, kjer sta zaradi povečanega pretoka poplavljali reki Medija in Sotla, hudo je bilo tudi na Ptuju, kjer je v nedeljo popoldne kar 12.000 uporabnikov ostalo brez električne energije.
?. Po podatkih uprave za zaščito in reševanje so nedeljska neurja prizadela  območja 46 občin, zaznali so več kot 100 dogodkov. Najhuje je bilo v občinah Zagorje, Rogatec in Rogaška Slatina, kjer sta zaradi povečanega pretoka poplavljali reki Medija in Sotla, hudo je bilo tudi na Ptuju, kjer je v nedeljo popoldne kar 12.000 uporabnikov ostalo brez električne energije.
TITLE: Isolation of Ashbya from bugs
AUTHORS: Amy Gladfelter

[DESCRIPTION]
Protocol to isolate Ashbya gossypii from milkweed and box elder bugs.

[STEPS]
2. To collect bugs put them in any sealable container. I like to use a 50 or 15ml conical with a few small holes in the lid but I've also used clean mason jars or the disposable coffee cups at conferences. In my experience live bugs work best. If possible record the species of insect, the location of collection, the date of collection, and the host plant if there is one. If you aren't sure you can always take pictures of the bug and the plant to identify later. t


8.   

1. Ashbya live in the mouths and digestive track of several species of insects, in particular the "true bugs"  aka Hemiptera. Examples of some of the species native to North Carolina. 
    
 
  

3. Prepare the plates. Use AFM with no selection. Top coat the plates with antibacterials, by adding 50ul of  Amp and Kan to the top of your plate, spreading it gently and allowing it to adsorb. It doesn't have to be these specific drugs, you just want to use something to cut down the number of bacterial colonies you get.     

4. Stick the bug into the 4deg for ~30min to chill it out.
5. Quickly sterilize a pair of forceps in a flame. Remove the bug from the container. Smush the bug's head and then mash it into the agar.  you can drag it around a little bit just try and make sure that it's contents end up on the agar. 

7. Place the plate at 25 or 30deg. Check on it every day. 
9. Once you get colonies pick each one to a new plate. I usually take no more than 4 from a single plate and by sequence they usually end up being the same (i.e. little to no diversity within a bug). Grow the plates until colonies are a few inches across and then make a mycelial stock and gDNA. you can determine species by sequencing with ITS primers or you can PCR the Whi3 locus and sequence that. 
6. example of the procedure.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol details how to create and merge Anvi'o profiles to view them using anvi-interactive. </p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is essentially the same as a standard ELISA protocol, with one major exception – all animal compounds must be avoided in buffers, etc. because they contain Neu5Gc, which will interfere with antibody activity and result in misleading data. Essentially, the glycoconjugate to be evaluated is immobilized on an ELISA plate and exposed to a primary antibody. The antibody provided in this kit has been shown to identify as little as 5 pmol of Neu5Gc per microgram glycoprotein, which is at or below the current detection limit for conventional DMB HPLC analysis. An advantage of the ELISA procedure is that it confirms the presence of Neu5Gc on the glycoconjugate of interest, but use caution, as it also reacts with solution/media that contains glycoconjugate.</p>

[GUIDELINES]
<p><strong> </strong></p>
<p><strong>Items required but not supplied:</strong></p>
<p> </p>
<ul>
<li>High-binding 96 well ELISA plate</li>
<li>Sodium carbonate-bicarbonate immobilization buffer 50mM, pH 9.5</li>
<li>Positive control target</li>
<li>Negative control target</li>
<li>PBS and PBS-T</li>
<li>Enzyme-conjugated secondary anti-chicken IgY antibody (HRP or AP)</li>
<li>Enzyme substrate</li>
</ul>

[STEPS]
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TITLE: Preparation of extracted DNA for long-read library prep
AUTHORS: Natalie Solonenko, Marie Burris

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details steps to prepare DNA for long-read library prep, including clean-up and shearing.</div></div>

[STEPS]
?. [Shear DNA]
Load at least 90ul of high molecular weight DNA into the top of a Covaris g-TUBE.  Loading less than 90ul will result in poorly sheared DNA.
?. [Shear DNA]
To shear DNA to 15kb, centrifuge at 4,700 rpm for 60 sec in an Eppendorf 5424 centrifuge with a 24 position rotor. Flip the g-TUBE upside-down and repeat this centrifugation.
You may also use Eppendorf model 5415 with a 24 position rotor. Using any other model or rotor size will result in DNA that is not sheared to the target size.
?. [Shear DNA]
To shear DNA to 20kb, centrifuge at 4,200 rpm for 60 sec in an Eppendorf 5424 centrifuge with a 24 position rotor. Flip the g-TUBE upside-down and repeat this centrifugation.
You may also use Eppendorf model 5415 with a 24 position rotor. Using any other model or rotor size will result in DNA that is not sheared to the target size.
?. [Shear DNA]
Recover DNA from the lid of the g-TUBE.
?. [Shear DNA]
Dilute a small aliquot to 2 - 50ng/ul and assess DNA size using Genomic DNA TapeStation.
If your DNA is less than 2ng/ul, proceed without checking DNA size.
?. [DNA clean-up]
Begin with high molecular weight DNA  (see "DNA Extraction for HMW DNA"). Assess the purity of your DNA using NanoDrop. If you have already done this and have the results available, you do not need to repeat it.
When assessing low concentration samples (
?. [DNA clean-up]
If your DNA has a visible peak at 260nm and your NanoDrop values are outside the following ranges -260/280: 1.7 - 2.1260/230: 1.8 - 2.2then your DNA needs clean-up!If your DNA does not have an easily visible peak at 260nm but has peaks elsewehere, then your DNA needs clean-up!
?. [DNA clean-up]
If your DNA is contaminated with phenol or other extraction reagents, perform an Ampure Bead clean-up (see "Ampure Bead Clean-up for High Molecular Weight DNA"). Elute in 100ul.
?. [DNA clean-up]
If your DNA is from an iron chloride precipitate resuspended with citrate buffer, clean up your DNA using the Qiagen DNeasy PowerClean Pro kit. Be sure to always mix gently by inversion or flicking, NOT vortexing or pipetting. Elute in 100ul.
?. [DNA clean-up]
Assess the conencentration of your DNA using Qubit, the purity using NanoDrop, and the size using Genomic DNA TapeStation.
?. [Shear DNA]
Proceed to "Long Read Viromics Amplification Library Preparation (VirION 2)".
TITLE: BioLogic Trim Keto Gummies Reviews - Fake Or Trusted?
AUTHORS: biologictrimketoreviews  

[DESCRIPTION]
BioLogic Trim Keto Gummies Reviews - Fake Or Trusted?


[STEPS]
1. BioLogic Trim Keto Gummies Reviews - Fake Or Trusted?
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Recent exploration has shown that numerous fake products are submerging the slimming lozenge request. Numerous of these don't meet their legal pledges and can beget adverse responses. 
Birth neat Keto Gummies This is the most recent and effective product, as it delivers ketosis fully deduced from the body's metabolic processes. 
What Is the Biologic Trim Keto Gummies? 
This new formula will help you see the benefits of the keto diet briskly than you allowed. We believe everyone should feel confident and comfortable in their skin, so we are always looking for products like this. This composition will give you all the details you need! We're confident that you'll love it if you order it now. For further information, continue reading this supplement review. 
There are numerous weight- loss fashions, but not all of them are of the same quality. Biologic Trim Keto Gummies capsules and other options are subject to our scrutiny to guarantee that our guests admit only the stylish. Numerous folks are too busy to probe motifs like this before asking for them. Smaller people know what to look for while conducting a background check after doing all the exploration and writing up the findings for you in one easy- to- understand piece. For a moment, you can know what this supplement accomplishes and how it heaps up against other options. You will learn about the factors and a slew of farther details! 
Biologic Trim Keto Gummies Constituents 
Ketone is an exogenous one, too. A substance known as BHB was used( short for beta- hydroxybutyrate). Still, you most probably understand why an exogenous ketone can be so salutary, but indeed if you do not, If you're well- clued in the keto diet. Before you make a request, we can give you with all the information you want. 

Official Website:- https://ipsnews.net/business/2022/05/26/biologic-trim-keto-gummies-reviews-is-acv-keto-gummies-real-or-fake/
https://biologic-trim-keto-gummies-reviews-fake-or-trusted.jimdosite.com/
https://educatorpages.com/site/dada30/pages/biologic-trim-keto-gummies-reviews-fake-or-trusted
TITLE: Guardian Blood Balance Australia
AUTHORS: health  

[DESCRIPTION]
Guardian Botanicals Blood Balance  Australia is, in my opinion a great product! This pill will assist your body to  repair itself. You can trust your judgement to make the right choice.

You  will live a happier, healthier life. It is a great treatment for  diabetes and high blood pressure. It is all-natural, non-toxic, and  completely safe! It will also address the root cause of your condition!

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Modified after V. Rich, 2/10/07: DNA extraction protocol from 25 mm 0.2 &mu;m filters &ndash; Low-throughput, using DNeasy columns (based on lab Steripak filter extraction protocol and the Suzuki et al. 2001 protocol)

[GUIDELINES]
<strong>Materials:&nbsp;</strong><br /><br />
<table>
<tbody>
<tr>
<td>Product</td>
<td>Vendor</td>
<td>Cat #</td>
</tr>
<tr>
<td>Sucrose, molecular biology grade</td>
<td>FISHER</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Lysozyme&nbsp;</td>
<td>Sigma</td>
<td>L7651-1G</td>
</tr>
<tr>
<td>Proteinase K, PCR grade</td>
<td>Roche</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>10% SDS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>96-100% EtOH</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Microcons 30 (now amicon)</td>
<td>Millipore</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Qiagen DNeasy Tissue Kit</td>
<td>Qiagen</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>

[STEPS]
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