eamag/protocols-with-steps huggingface dataset:
TITLE: General Fungal DNA Extraction
AUTHORS: Angie Macias, Matthew T Kasson, Brian Lovett

[DESCRIPTION]
This is a routine protocol for extracting DNA from various fungi. This extraction method is suitable for follow-up molecular work such as PCR amplification.

[STEPS]
SECTION: Before you begin
1. Turn on hot water bath, set to 65 °C.

SECTION: Before you begin
2. Pull two Eppendorf 1.5 mL centrifuge tubes per sample.

SECTION: Before you begin
2.1. Label both sets of tubes with (short) sample names.
SECTION: Before you begin
2.2. Label one tube set for each sample with an "I" for  .
  

SECTION: Before you begin
3. Add 200 µL of   (or () to tubes without "I".

SECTION: Before you begin
4. Add 600 µL of   to tubes labeled with "I".

SECTION: Before you begin
5. Place tube with   into 65 °C water bath.

SECTION: Extraction Protocol
6. Sterilize some metal scrapers with flame and 95 % (v/v)   .

SECTION: Extraction Protocol
7. Add 1/2 pea-sized amount of fungal tissue (young hyphae) to each tube containing  .
SECTION: Extraction Protocol
7.1. Flame-sterilize and cool scrapers between samples.
SECTION: Extraction Protocol
8. Macerate each sample with a new, sterile micropestle until tissue is homogenous.
SECTION: Extraction Protocol
10. Add tubes to a floating rack to allow samples to incubate directly in 65 °C water bath for 30 min.

SECTION: Extraction Protocol
11. Remove samples and vortex for 3 s before returning to 65 °C water bath for 30 min.

SECTION: Extraction Protocol
12. Remove samples and allow them to cool on the bench for 5 min.

SECTION: Extraction Protocol
13. Add 200 µL of   to each tube and vortex for 10 seconds.

SECTION: Extraction Protocol
14. Centrifuge samples for 3 min at 14.000 rpm.

 

SECTION: Extraction Protocol
15. Using a P1000 micropipette, transfer supernatant to each tube containing   and gently mix by inversion.
 


SECTION: Extraction Protocol
16. Centrifuge for 1 min at 14.000 rpm.

SECTION: Extraction Protocol
17. Carefully pour off the supernatant into waste container.
 

SECTION: Extraction Protocol
18. Add 600 µL of 70 % (v/v)   to each tube and mix gently by inversion.

SECTION: Extraction Protocol
19. Centrifuge for 1 min at 14.000 rpm.

SECTION: Extraction Protocol
20. Repeat Step 16.
SECTION: Extraction Protocol
21. Open and invert tubes onto a clean paper towel.
 

SECTION: Extraction Protocol
22. Add 100 µL of warmed   to each tube.

SECTION: Extraction Protocol
23. Store fully-labeled tubes in a box (not a tube rack) in the -20 °C freezer.

SECTION: Extraction Protocol
11.1. Place a sufficient aliquot of   in water bath to warm for Step 21.

SECTION: Extraction Protocol
9. Add 400 µL of   (to 600 µL total volume added).
SECTION: Extraction Protocol
7.2. Alternatively, pellet a pea-sized amount of mycelium grown in liquid culture and transfer to each tube.
TITLE: IPA assembly for Hifi PacBio reads
AUTHORS: a.kharabianmasouleh , Priyanka Sharma, Robert Henry

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is regarding the assembly of plant genomes using HiFi data (PacBio Sequel II) using Improved Phase Assembly (IPA) tool.</div></div>

[STEPS]
?. Steps to generate the genome assembly of different plant species using IPA (Improved phased assembly):  1) ccs.bam files generated by PacBio Sequel II (Hifi) platform for all the plant species were used to assemble the genome using IPA tool.2) IPA was installed 2) IPA was initiated using Conda environment
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p> </p>
<ul>
<li><em>B. saltans</em> cells were electroporated using a square-wave electroporator (Nepa21, Bulldog Bio, Inc.) using one poring pulse of 200 volts with a pulse duration of 25 ms and five transfer pulses of 60 volts with a pulse duration of 99 ms, with plasmid targeting the 18S region (18S-GFP). A schematic representation of the plasmid, the target locus and the expected site of integration into the <em>B. saltans</em> genome is shown in Figure 1.</li>
</ul>
<p> </p>
<ul>
<li>Electroporated cells were selected with 1 µg/ml of G418, added 24 hours after electroporation. Cells were washed and subcultured into fresh selection medium every 3-4 days. G418 resistant cells started to emerge 7-9 days post-electroporation.</li>
</ul>
<p> </p>
<ul>
<li>Cells were processed for genotyping analysis to confirm plasmid integration 3 weeks post-electroporation. DNA was extracted from pools of transfected and wild cells using the Qiagen DNeasy Blood &amp; Tissue kit.</li>
</ul>
<p> </p>
<ul>
<li>PCR analyses were used to characterize the 18S-GFP tagging using 6 sets of PCR primers, as shown in Figure 1 C.</li>
</ul>
<p> </p>
<ul>
<li>Gel electrophoresis image (Figure 2) showing the amplified PCR products at the expected sizes.</li>
</ul>
<p> </p>
<ul>
<li>Amplified PCR#1 with primer sets Ribo_tag_forward &amp; GFP reverse (800 bp)</li>
<li>Amplified PCR #2 with primer sets Neo_forward &amp; Ribo_tag_reverse (1000 bp)</li>
<li>Amplified PCR #3 with primer sets TubR &amp; IG forward ( 3 bands)</li>
<li>Amplified PCR #4 with primer sets Tub_forward &amp; IG reverse (3 bands)</li>
<li>Amplified PCR #5 with primer sets Ribo_tag_forward &amp; Ribo_tag_reverse (Wild (C) cells band at 350 bp, transfected cells S1 and S2 two bands, 350 bp and 2800 bp)</li>
</ul>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p>Figure 1: Schematic representation of the (A); the 18S- GFP plasmid; (B) Ribosomal operon in <em>B. saltans</em> genome (C); and the expected site of plasmid integration in <em>B. saltans</em> genome through homologous regions 1 and 2 (HR1, HR2).</p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p>Figure 2: Agarose gel electrophoresis image of the amplified PCR products for <em>B. saltans</em> cells transfected with 18S-GFP Cassette (S1 and S2) and the wild type cells (C). The primers sets and the expected product sizes are mentioned above in the text.</p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p>Figure 3: <em>B. saltans</em> cells transfected with 18S-GFP tagging cassette. On the left: a light microscopy image showing the <em>B. saltans</em> cells (around 4 cells at different level). On the right: GFP fluorescent signal detected in the transfected cell. Scale bar 10 μm.</p>
<p> </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This mock protocol is a fun description of the steps one might undertake to train for a one-way mission to the planet Mars.  The One-Way mission concept is gaining popularity due to it's simpler approach and lower cost.  Rather than expend effort to bring back the Marstronauts, the plan calls for leaving them there and ultimately colonizing the red planet.<br /><br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/ctvdy6w.jpg" data-src="https://s3.amazonaws.com/pr-journal/ctudy6w.jpg" data-ofn="lone astronaut.jpg" />

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Create a script that is the reverse of ‘cat’ in that it will print the lines from a given input file in reverse order:</p>
<pre><code>$ cat input.txt
foo
bar
baz
quux
$ ./tac.pl6 input.txt
quux
baz
bar
foo
</code></pre>
<p>The script should accept a single unnamed “input” argument that should be a file. If provided no arguments or an argument that is not an existing file, the script should produce a “usage” statement:</p>
<pre><code>$ ./tac.pl6
Usage:
  ./tac.pl6 &lt;input&gt;
$ ./tac.pl6 this-does-not-exist
Usage:
  ./tac.pl6 &lt;input&gt;
</code></pre>
<p>If given an input that does not exist, it should print a usage statement.</p>
<p>A passing test suite should look like this:</p>
<pre><code>$ make test
./test.pl6
1..3
ok 1 - No args gives usage
ok 2 - Bad arg gives usage
ok 3 - Correct output
</code></pre>
<p>If you “make jabber,” you should see this:</p>
<pre><code>  And the mome raths outgrabe.
All mimsy were the borogoves,
  Did gyre and gimble in the wabe;
'Twas brillig, and the slithy toves

  He chortled in his joy.
O frabjous day! Callooh! Callay!'
  Come to my arms, my beamish boy!
"And, has thou slain the Jabberwock?

  He went galumphing back.
He left it dead, and with its head
  The vorpal blade went snicker-snack!
One, two! One, two! And through and through

  And burbled as it came!
Came whiffling through the tulgey wood,
  The Jabberwock, with eyes of flame,
And, as in uffish thought he stood,

  And stood awhile in thought.
So rested he by the Tumtum tree,
  Long time the manxome foe he sought --
He took his vorpal sword in hand:

  The frumious Bandersnatch!"
Beware the Jubjub bird, and shun
  The jaws that bite, the claws that catch!
"Beware the Jabberwock, my son!

  And the mome raths outgrabe.
All mimsy were the borogoves,
  Did gyre and gimble in the wabe:
'Twas brillig, and the slithy toves

JABBERWOCKY -- Lewis Carroll</code></pre>

[STEPS]
?. 
?. 
?. 
?.
TITLE: Colony PCR
AUTHORS: Zhujun Wei

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This PCR method can be used to screen for inserted target genes or DNA sequencing analysis.</div></div>

[STEPS]
?. Pick colonies as the template for colony PCR. The number picked for each plate depends on the difference between the positive and negative controls.  AB12×high Taq Master Mix (Enzyme)5 μl2Template1 μl3Forward Primer (10 μM)0.4μl4Reverse Primer (10 μM)0.4 μl5ddH2O3.2 μlFill the rest with water.
AB12×high Taq Master Mix (Enzyme)5 μl2Template1 μl3Forward Primer (10 μM)0.4μl4Reverse Primer (10 μM)0.4 μl5ddH2O3.2 μl
?. Test digest performed and products analysed using agarose gel electrophoresis to confirm if correct construct was present.
?. Use colony PCR to enlarge colony numbers. Then only the positive clones were mini-prepped.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>BiMat: a MATLAB package to facilitate the analysis of<br />bipartite networks.<br /> <br />Extensive documentation, code, and worked examples available on:<br /> <br />http://bimat.github.io</p>
<p> </p>
<p>The datasets included with the package include phage-bacterai infection networks. </p>

[STEPS]
?.
TITLE: Digestion for BioBrick Assembly Kit (E0546)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
This protocol explains methods for assembling multi-compontent genetic systems using BioBrick® parts. 

[BEFORE_START]
The BioBrick® Assembly Kit was developed in partnership with Ginkgo BioWorks. What follows is an abbreviated set of protocols for the use of the BioBrick® Assembly Kit (to assemble an Upstream Part with a Downstream Part into a Destination Plasmid). For more details and for technical questions, please see here.

[GUIDELINES]
The BioBrick® Assembly Kit provides a streamlined method for assembly of BioBrick parts into multi-component genetic systems. BioBrick parts are DNA sequences that encode a defined biological function and can be readily assembled with any other BioBrick part. The process for assembling any two BioBrick parts is identical and results in a new composite BioBrick part.

The BioBrick Assembly Kit contains EcoRI-HF®, XbaI, SpeI, PstI, T4 DNA Ligase and NEBuffer 2.1.
 

BioBrick Assembly Overview

[STEPS]
1. Digest Upstream Part with EcoRI-HF® and SpeI:
 

2. Digest Downstream Part with XbaI and PstI:
 

3. Digest the Destination Plasmid with EcoRI-HF® and PstI:  
 

 

4. Incubate all three restriction digest reactions at 37 °C for 10 min.
5. Heat inactivate at 80 °C for 20 min.
TITLE: Standard Operating Procedure for karyotyping non-model organisms
AUTHORS: Ana Riesgo, marietaconejero, C.DiNizo, Jonas Astrin, cira.martinez, Tomás Marqués-Bonet

[DESCRIPTION]
The SOP we developed for karyotyping non-model organism is a combination of several
protocols, with modifications (see references in document). Metaphases obtained from cell cultures have a higher
quality compared to the ones from tissues, since cell division can be observed and controlled, and the chromosomes are more distended. It requires, however, development of a cell culture, which is not trivial for many taxa. It also needs adjustments in relation to the time of action of colchicine in different taxa and modifications of buffers to maintain osmolarity when dealing with marine/freshwater organisms. Overall, karyotyping methods from cell cultures are well established and have been done routinely at our labs for mammals, birds, reptiles, and fishes. For the latter two groups, it is more time consuming since the cells must be exposed to
colchicine for longer periods (minimum 3h until overnight).  Karyotyping tissues can prove more challenging due to variation in penetration of colchicine, timing of the incubation in the different reagents, which also needs to be adjusted for every target group, among other things. In addition, in some cases, it requires the injection of colchicine in vivo, which makes the process more bureaucratic since it requires obtaining local permits, approval from local ethics committees, and in some cases the presence of a veterinarian. Furthermore, it is difficult to control cell division and consequently the
metaphase index is lower compared to karyotypes from cells. Even though cell culture is the best source of karyotyping (with the advantage of being permanently stored and reused), tissue karyotyping has proven to be a very useful
alternative in cases where cells are difficult to obtain (some fishes, amphibians, snakes and invertebrates). Furthermore, cell culture karyotypes are more subject to mutation if many passages occur. 

[STEPS]
TITLE: Respiratory rate (microCT)
AUTHORS: Núria Peñuelas

[DESCRIPTION]
Respiratory rate (microCT) measurement in mice

[STEPS]
1. For the scanning procedure, anestethize the animals with isoflurane (5% induction phase, 1% maintenance). Set the air flow to 0.8l/min.
2. Define parameters for image acquisition (field of view: 73 mm, acquisition time: 2 min, current voltage:70 mV, and amperage:200 uA).
3. Determine respiration frequency using microCT measurements acquired with Quantum FX imaging system (Perkin Elmer. 940 Winter St. Waltham, Massachusetts. EEUU).
TITLE: Striatal sections immunofluorescence and analysis
AUTHORS: Chuyu Chen

[DESCRIPTION]
Previous studies have shown that the phosphorylation of ribosomal protein S6 (p-rpS6) at S235/236 residues increases in iSPNs in response to haloperidol activation. This phosphorylation is cAMP/PKA dependent, which is the canonical pathway inhibited by D2R antagonism. We therefore explored whether LRRK2 inhibition interferes with this haloperidol-mediated phosphorylation event.

[STEPS]
SECTION: Immunostaining
1. Drd2-eGFP mice crossed with LRRK2-GS KI and control littermates were treated as indicated and perfused with 50 ml of PBS
SECTION: Immunostaining
2. Mice were perfused with 4% paraformaldehyde in PBS. 
SECTION: Immunostaining
3. Brains were dehydrated with 30% sucrose in PBS for 48hrs and cut coronally (30 μm) by cryostat
SECTION: Immunostaining
4. Striatal sections were incubated with 5% goat serum in 0.2% Triton X-100 for 2 hrs
SECTION: Immunostaining
5. the sections were incubated in the same solution overnight at 4 °C with the primary antibodies anti-GFP (1:1000, Invitrogen) and anti-phospho-S6 Ribosomal protein (Ser236/236) (1:300, Cell Signaling Technology)
SECTION: Immunostaining
6. Sections were washed with PBS for 5mins at RT
SECTION: Immunostaining
7. Repeat the wash with fresh PBS
SECTION: Immunostaining
8. Incubate with the secondary antibodies Alexa Fluor‱ 488 and Alexa Fluor‱ 647 (both 1:600, Invitrogen) for 3 hrs.
SECTION: Immunostaining
11. Sections were mounted on ProLong Diamond Antifade Mountant
SECTION: Immunostaining
9. Sections were washed with PBS for 5mins at RT
SECTION: Immunostaining
10. Repeat the wash with fresh PBS
SECTION: Confocal imaging
12. Fluorescence images were obtained with Nikon A1R microscope and were acquired with a 20x objective at 1,024 x1,024 pixel resolution
SECTION: Confocal imaging
12.1. Stitched images of the whole striatum were automatically acquired with Nikon Element
SECTION: Image analysis
13. GFP and phospho-S6 Ribosomal protein signal were measured using Imaris 10.1 software
SECTION: Image analysis
14. Surface rendering function was used to segment Drd2-eGFP cells
SECTION: Image analysis
14.1. Background subtraction was enabled
SECTION: Image analysis
14.2. The diameter of the largest sphere was set at 15 μm and automatically thresholded
SECTION: Image analysis
14.3. Smooth surface was set to 1.23 μm
SECTION: Image analysis
15. Mean intensity of phospho-S6 Ribosomal protein within Drd2-eGFP surface above 2 times of average phospho-S6 Ribosomal protein channel mean intensity was considered positive
SECTION: Image analysis
14.4. Segments were filtered with an area ranging between 100 μm2 and 6,000 μm2
TITLE: 0.5M EDTA, pH 8.0
AUTHORS: Adrien Assie, Buck Samuel

[DESCRIPTION]
EDTA recipe

[STEPS]
1. •	Start with 700 mL water
2. •	Add ~60 g of NaOH
3. •	Allow to cool back to room temperature (can speed in room temp water bath)
4. •	Add 146.12 g EDTA
5. •	Adjust pH to 8.0 with 5-10 M NaOH
6. •	Add water to 1000 mL
7. •	Autoclave
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The process of developing transient transfection protocols in <em>P. caudatus</em> involved initial testing and determination of proper electroporation buffers and parameters (voltage strength, pulse duration and number) to maximize cell viability. Our results suggest that the type of electroporation buffer is critical for maintaining cell viability throughout the experiment and is essential for determining the optimum electric field range.</p>

[STEPS]
?. 
?. 
?.
TITLE: Skimmed Milk Flocculation Technique for Waste Water
AUTHORS: Dilip Abraham, Venkata Raghava Mohan, gkang 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The direct examination of waste water is difficult due to low and fluctuating concentrations of microorganisms and because concentration procedures are usually organism and/or matrix specific and most techniques have high or unknown variability parameters. One-step skimmed milk flocculation (SMF) has been proposed as an efficient method to concentrate viruses in all types of environmental water matrices such as river water, seawater, groundwater and wastewater. The organic flocculation procedure is easy and fast to be carried out; it requires a simple laboratory infrastructure, thus eliminating costly equipment, in addition to the low cost of the inputs necessary to be performed. The aim of this study is to perform skimmed milk flocculation procedure in order to detect SARS-CoV-2 recovery from the waste water.</div></div>

[STEPS]
?. [Skimmed Milk Flocculation]
For virus concentration assay, collect a total of  waste water, and divide into two aliquots ().
250 mL
100 mL
?. [Skimmed Milk Flocculation]
To each  sample, add  5% pre-flocculated skim milk (e.g., add  for  of sample) and adjust the - by the addition of  HCl.
100 mL
1 mL
2.5 mL
250 mL
?. [Skimmed Milk Flocculation]
Place the samples on a horizontal shaker and incubate in agitation at  for  at  to allow the floc formation.
Centrifuge: 200 33
4 Room temperature
?. [Skimmed Milk Flocculation]
After incubation, allow the flocculants to sediment by centrifugation at  for  at  using a swinging bucket rotor.
Centrifuge: 3500 34
4 °C
?. [Skimmed Milk Flocculation]
Remove the supernatant carefully from the tube without disturbing the bottom and suspend the pelleted virus with the following buffer.
?. [Skimmed Milk Flocculation]
Use 1 set of pellet for RNA extraction using the QIAamp viral RNA mini kit by suspending it with the lysis buffer provided in the kit.
?. [Skimmed Milk Flocculation]
Dissolve another aliquot in  of sterile 1x PBS () and vortex both the suspension for - at maximum speed to completely dissolve the pellet.
1.5 mL
?. [Skimmed Milk Flocculation]
Store the final viral concentrates at - until further processing.
80 °C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Wheat—a staple food crop of many countries, has always been a target for improving its resilience to biotic and abiotic stresses. Among these, rust diseases are the most detrimental in depressing wheat production. In the present study, chemical mutagen was used to induce mutations in a wheat variety NN-Gandum-1 (moderately resistant to leaf and yellow rust) for improving resistance to the disease as well as studying function of genes conferring resistance to the disease. In the present investigation, 0.8% EMS dose was found optimum for supporting 45-55% germination of NN-Gandum-1. A total of 3,634 M<sub>2</sub> fertile plants were produced from each of the M<sub>1</sub> plant. Out of these, 33 (0.91 %) and 20 plants (0.55%) showed absolute resistance to leaf and yellow rust, respectively. While 126 (3.46%) and 127 plants (3.49%) exhibited high susceptibility to the leaf and yellow rust, respectively. In M<sub>4</sub> generation, a total of 11 M<sub>4</sub> lines (nine absolute resistant and two highly susceptible) and one wild type were selected for NGS-based exome capture assay. A total of 104,779 SNPs were identified that were randomly distributed throughout the wheat sub genomes (A, B and D). Maximum number of mutations were found in introns. Highest number of SNPs were detected in chr.2B (14,273 SNPs). Mutation density was one mutation per 20.91 Mb. Highest mutation frequency was found in chr.7D (1/2.83 Mb) and the lowest in chr.2D (1/85.17 Mb). Out of the detected mutations, 101 SNPs were selected using priority analysis criteria. Out of these, SNPs detected in <em>Lr21</em> were selected for further analysis. The SNP identified in chimeric allele (<em>Lr21</em>) of a resistant mutant (N1-252) was located in NBS domain of chr.1BS at 3.4 Mb that possibly activated the function of chimeric allele. Through computational analysis, it was demonstrated that identified SNP substituted the glutamic acid with alanine—resulted in altered protein structure. It was concluded that induced mutations are extremely helpful not only in generating novel genetic resource but can also exploited for getting insight into the important biological circuits of different traits of complex genomes like wheat.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Describes collection for the following:</p>
<p> </p>
<p>Domoic acid (particulate and dissolved)*</p>
<p>Chlorophyll</p>
<p>DNA</p>
<p>Nutrients: NH4, urea, N, P, Si</p>
<p>Flow cytometer counts (Synecocccus, pico-eukaryotes, heterotrophic bacteria)</p>
<p>Whole water/net tow archive</p>
<p>Whole cell probe counts (P. australis, P. multiseries, Alexandrium)</p>
<p> </p>
<p>*Filters are also used for HPLC pigment analysis</p>
<p> </p>

[STEPS]