eamag/protocols-with-steps huggingface dataset:
TITLE: Cryo_preservation_Synechocystis_PCC_6803
AUTHORS: maurice.mager1808

[DESCRIPTION]
Cryopreservation protocol used during the interlaboratory study published by Mager et al. 2023.

[STEPS]
SECTION: Precultures for Cryo conservation of Synechocystis PCC 6803
1. Starting from a BG11 agar plate with your strain of interest, inoculate a BG11 liquid culture

 

SECTION: Cryo conservation of Synechocystis PCC 6803
2. Centrifuge cells at 12.000 g (fixed angle rotor) for 15 min and wash in the same volume of fresh BG11 medium
SECTION: Inoculating a culture from Cryo conserved Synechocystis PCC 6803 cells
3. Use 330 µL of prepared cryoconserved cells to inoculate a 100 ml flask containing 10 mL of  BG11 medium

SECTION: Precultures for Cryo conservation of Synechocystis PCC 6803
1.1. Grow the strain of interest with your settings of choice. 

 


SECTION: Precultures for Cryo conservation of Synechocystis PCC 6803
1.2. Grow the cells to an OD730 of 2.5-3
SECTION: Cryo conservation of Synechocystis PCC 6803
2.1. Centrifuge cells at 12.000 g (fixed angle rotor) for 15 min and resuspend in 10% of the initial volume with BG11 and 15% (v/v) final concentration glycerol


 

SECTION: Cryo conservation of Synechocystis PCC 6803
2.2. Aliquot cells in tubes to 350µl each
SECTION: Cryo conservation of Synechocystis PCC 6803
2.3. Optionally, freeze cells in liquid nitrogen. Store cells at -80°C. 


 

SECTION: Inoculating a culture from Cryo conserved Synechocystis PCC 6803 cells
3.1. Cultivate for 48 hours at your settings of choice
TITLE: Advances in Hydrogen-deuterium Exchange Mass Spectrometry-A New Method for Protein Structure Analysis
AUTHORS: 181830691 , fe

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Hydrogen - deuterium exchange mass spectrometry is to study the spatial conformation of proteins. It is widely used in the study of protein structure and dynamic changes, the discovery of protein interaction sites, the identification of protein epitopes and active sites. With the continuous development of hydrogen-deuterium exchange mass spectrometry, it is becoming an important approach for structural biologists and biological drug development.</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div>
<p>In the study we have used a standard Thermo Fisher Scientific Sanger sequencing protocol which can be found under this link:</p>
<p> </p>
<p>http://www.ramaciotti.unsw.edu.au/wp-content/uploads/2016/08/sequencing_handbook_FLR.pdf</p>
</div>

[STEPS]
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
NS-Forest is an alogrithm that determines the minimum set of genes that are necessary and sufficient to define a cell type cluster derived from single cell RNAseq expression data. 

Development and stable releases can be found at : 

https://github.com/JCVenterInstitute/NSForest

[STEPS]
SECTION: Pre-analysis data preparation
?. 
SECTION: Input parameters
?. 
SECTION: Core algorithm description
?. 
SECTION: Results reporting
?.
TITLE: U Mass - Exercise model
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">In-cage free running wheel is used to voluntarily induce exercise in awake mice. Exercise affects insulin sensitivity and glucose metabolism.</div></div>

[STEPS]
?. Place running wheel inside mouse home cage.
?. Monitor mouse health.
TITLE: Alpha-synuclein immunochemistry on STC-1 cells using DAB
AUTHORS: Michael J Hurley

[DESCRIPTION]
This protocol describes how to visualise alpha-synuclein in STC-1 cells by DAB immunohistochemistry. It also works for other antibodies (e.g. 5-HT, CCK, GLP).

[STEPS]
SECTION: Alpha-synuclein immunochemistry on STC-1 cells using DAB
5. Thaw frozen 24-well plates containing fixed cells grown on glass coverslips
SECTION: Alpha-synuclein immunochemistry on STC-1 cells using DAB
6. Wash cells with PBS 5 min

SECTION: Alpha-synuclein immunochemistry on STC-1 cells using DAB
7. Quench with 0.3% H2O2 5 min

SECTION: Alpha-synuclein immunochemistry on STC-1 cells using DAB
8. Wash cells with PBS 2 x 5 min

SECTION: Alpha-synuclein immunochemistry on STC-1 cells using DAB
9. Block with 10 % goat serum in PBS containing 0.005 % Triton™ X-100 and 0.05 % thimerosal60 min

SECTION: Alpha-synuclein immunochemistry on STC-1 cells using DAB
10. Incubate with rabbit anti-alpha-synuclein (1:500) (Abcam, ab212184) in 10 % goat serum containing 0.005 % Triton™ X-100 and 0.05 % thimerosal (block buffer) 1200 min

SECTION: Alpha-synuclein immunochemistry on STC-1 cells using DAB
11. Wash with PBS  4 x 5 min

SECTION: Alpha-synuclein immunochemistry on STC-1 cells using DAB
12. Incubate with biotinylated goat anti-rabbit IgG (1:500) (Stratech, 111-065-144-JIR) in 2 % goat serum containing 0.005 % Triton™ X-100 and 0.05 % thimerosal (antibody buffer) 120 min

SECTION: Alpha-synuclein immunochemistry on STC-1 cells using DAB
13. Wash with PBS  4 x 5 min

SECTION: Alpha-synuclein immunochemistry on STC-1 cells using DAB
14. Incubate with 1:250 streptavidin-HRP (Roche, 11089153001) for 1 hour in antibody buffer60 min

SECTION: Alpha-synuclein immunochemistry on STC-1 cells using DAB
15. Wash with PBS 6 x 5 min

SECTION: Alpha-synuclein immunochemistry on STC-1 cells using DAB
16. Visualise staining by incubation with PBS containing 0.05 % (w/v) 3,3′-Diaminobenzidine/0.015 % (v/v) H2O2/0.05 % (w/v) nickel ammonium sulphate for ≤ 3 min

SECTION: Alpha-synuclein immunochemistry on STC-1 cells using DAB
17. Rinse for > 5 min in running tap water

SECTION: Alpha-synuclein immunochemistry on STC-1 cells using DAB
18. Remove coverslips from wells and mount on microscope slides with Hydromount™ (National Diagnostics)
SECTION: Alpha-synuclein immunochemistry on STC-1 cells using DAB
1. Grow STC-1 cells on 12 mm glass coverslips treated with poly-D-lysine or Geltrex™. 
SECTION: Alpha-synuclein immunochemistry on STC-1 cells using DAB
2. Fix cells with 4 % paraformaldehyde 15 min

 

SECTION: Alpha-synuclein immunochemistry on STC-1 cells using DAB
3. Wash cells with PBS 2 x 5 min

SECTION: Alpha-synuclein immunochemistry on STC-1 cells using DAB
4. Aspirate and discard PBS and freeze cells < -20 °C
TITLE: Intestinal transit time
AUTHORS: Núria Peñuelas

[DESCRIPTION]
Intestinal transit time measurement in mice

[STEPS]
1. Prepare red dye (Carminered dye, Sigma #C1022) in 0,5% methylcellulose (Sigma #M7027).
2. Take the animal and administer the red dye by oral gavage.
3. After oral gavage, individualize the animals in separate boxes without bedding.
4. Monitor the animal boxes for a total time of 4h.
5. Register the time when the animals defecate the first dyed fece (stained red).
6. Represent the time until first dyed fece as the transit time of the animals.
TITLE: Calf stretching
AUTHORS: Masood  Khan

[DESCRIPTION]
This time course study aimed to assess the acute effects of static stretching (SS) of different durations on the isometric maximum voluntary contraction force (MVCF) of the calf muscle. Ten male participants participated in three pretest-posttest experimental trials: SS for 2-minutes (SS2), 4-minutes (SS4), and 8-minutes (SS8). MVCF was measured before, immediately after, at 10- and 20-minutes post-stretch intervals. SS2, SS4, and SS8 did not change the MVCF at 0-, 10- and 20-minutes post stretching intervals.

[STEPS]
TITLE:        ELISA for quantification of human properdin in serum or plasma.
AUTHORS: Angel  Justiz-Vaillant, Belkis Ferrer-Cosme

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

[STEPS]
?. An anti-human properdin coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum or plasma. Human properdin present in the serum or plasma binds to antibodies adsorbed into the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-properdin antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-properdin antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3,3',5,5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 20 min.
?. A colored product is formed in proportion to the quantity of human properdin present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human properdin standard dilutions and the human properdin sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
In this protocol, we will merge the 20 profiled metagenomes in a table of relative abundances and visualize the table with a heatmap.

[BEFORE_START]
REQUIREMENTS: the <a href="http://matplotlib.org/" target="_blank">matplotlib</a> python library installed.

[GUIDELINES]
The commands reported in this protocol can be retrieved as a <a href="https://bitbucket.org/nsegata/metaphlan/wiki/2_metaphlan_merge_and_visualize.sh" target="_blank">bash script</a>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Here we present an efficient way to grow Tp colonies inside a 0.25% superclean agar matrix. This protocol could possibly be applicable to other marine microeukaryotes that are problematic to grow on fully solid support.</p>

[BEFORE_START]
<p><strong>1. Tp L1</strong></p>
<p> </p>
<p>Prepare L1 medium with Aquil salts as described by Karas et al. (2015). After sterile filtration, add 5 mL 30 g/L Na2SiO3 x 9H2O per 1L L1. More details on exact medium preparation can be found <a href="http://www.openwetware.org/images/2/2f/JT_L1_Aquil_generic.pdf" target="_blank">here</a>. This medium is referred to as 'Tp L1' in the protocol.</p>
<p> </p>
<p>Reference:</p>
<p>Karas, B.J. et al. (2015) Designer diatom episomes delivered by bacterial conjugation. Nature Communications. 6, 692-695.</p>
<p> </p>
<p><strong>2. Superclean agar </strong></p>
<p> </p>
<p>Prepare according to Waterbury &amp; Willey (1988). Excerpt:</p>
<p> </p>
<p>'100 g of agar is washed by stirring with 3 liters of double-distilled water in a 4-liter beaker. After 30 min of stirring, the agar is allowed to settle, the wash water is siphoned off, and the agar is filtered onto Whatman F4 filter paper in a Buchner funnel. This procedure is repeated once more or until the filtrate is clear. The agar is then washed with 3 liters of 95% ethanol followed by a final 3-liter wash with analytical grade acetone. The agar is then dried at 50 °C in glass baking dishes for 2-3 days and stored in a tightly covered container.' </p>
<p> </p>
<p>Use bacteriological agar from Sigma --&gt;<a href="http://www.sigmaaldrich.com/catalog/product/sial/a5306?lang=en&amp;region=US" target="_blank"> LINK</a>.</p>
<p> </p>
<p>Reference:</p>
<p>Waterbury, J.B. &amp; Willey, J.M. 1988 Isolation and Growth of Marine Planktonic Cyanobacteria. Methods in Enzymology, vol. 167, pp. 100-105. </p>

[STEPS]
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TITLE: Membrane challenge
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Membrane Stress Challenge Protocol</div></div>

[STEPS]
?. [Protocol]
Prepare a 10 ml overnight of OG1RF in BHI. Grow at 37°C
?. [Protocol]
Prepare dilution tubes with 0.9% saline - autoclave dilution tubes for 12 minutes
?. [Protocol]
In a.m., dilute overnight to b 0.01 in x of BHI + 1.5mM CaCl2Prepare BHI +1.5mM CaCl2: Depending on how much BHI you need, you can take a 250 mL flask, add 100 ml BHI, and 150μl of 1 M CaCl2.  This can be served for blanking and resuspending cells at the end.
Cell growth conditions for long term supplementationAt the onset of growth (0.01 OD600) add your fatty acid supplements.Fatty acid supplementation may impact generation time – so start cultures accordingly
?. [Protocol]
Once cells about 0.3, harvest10ml of cell (can spin in plastic)
Cell growth conditions for short-term supplementationGrow cells in the absence of fatty acid supplements.Once cells reach ~0.225-0.25 spike in fatty acid supplementIf investigating 2 different fatty acid supplements – grow 25 mL of BHI and then divide into two aliquots with 10 mL – increase the initial quantity of BHI accordingly.Incubate for 30 minutes at 37°C
?. [Protocol]
Wash cells in 10 ml 1x PBS
?. [Protocol]
Resuspend cels in 10 ml BHI = 1.5 mM CaCl2
?. [Protocol]
Transfer 3 ml to a glass tube.
?. [Protocol]
Add membrane stress agentDaptomycin to be 30μg/ml (should be 90μl of 1 mg/ml stock)       - Be sure to check on the concentration you need for your experiment. 30 is just an example.SDS to be 0.05% (stock is 10% SDS – 15ul into 3 ml)       - Be sure to check on the concentration you need for your experiment. 30 is just an example.
?. [Protocol]
At times 15, 30, 60 post-membrane stressors, make serial dilutions that can range from 10-1 to 10-7. Consider a pilot to determine the necessary dilution ranges. Plate the dilutions.
?. [Protocol]
Incubate plates overnightat 37°C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div>
<div>
<div>This is our standard brain tissue dissociation protocol for cell sorting (<a href="https://www.humancellatlas.org/" target="_blank" rel="noopener noreferrer">Human Cell Atlas</a>).</div>
</div>
</div>

[GUIDELINES]
<p><strong>NOTE</strong>: A critical component of this protocol requires proper handling of the tissue donation starting immediately at the time of autopsy. The overall sample viability depends significantly on minimizing the post-mortem interval (PMI), immediately transferring tissue into the appropriate cold medium supplemented with antibiotics/antimycotics, keeping the sample on <strong>wet</strong> ice throughout transportation, and maintaining sterile technique throughout the protocol.</p>
<p> </p>
<p><strong>The protocols workflow is as follows:</strong></p>
<p> </p>
<p>1. Obtaining tissue samples (Steps 1-9)</p>
<p>2. Preparation for sample processing (Steps 10-15)</p>
<p>3. Mechanical dissociation (Steps 16-21)</p>
<p>4. Enzymatic dissociation (Steps 22-32)</p>
<p>5. Sucrose gradient centrifugation (Steps 33-39)</p>
<p>6. ACK red blood cell lysis (Steps 40-44)</p>
<p> </p>
<p><strong>Thumbnail Image:</strong> </p>
<p><a href="https://www.humancellatlas.org/areas-of-impact" target="_blank" rel="noopener noreferrer">https://www.humancellatlas.org/areas-of-impact</a></p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
April 2000 chp<br /><br />Phage are best purified by a CsCl gradient. This is separated by density not sedimentation as in a sucrose gradient.<br /><br />This is a protocol for P22 like phage not T4 with delicate tail fibers.

[GUIDELINES]
This protocol is part of a larger collection of Cesium-Chloride related protocols. This is number (2) of (4):<br />1. <a href="https://www.protocols.io/view/Cesium-Chloride-gradients-and-DNA-Extraction-Virus-c7dzi5" target="_blank">Cesium Chloride Gradients</a><br />2. <a href="https://www.protocols.io/view/CsCl-Step-Gradient-to-Purify-Phage-c4zyx5" target="_blank">CsCl Step Gradient to Purify Phage</a><br />3. <a href="https://www.protocols.io/view/DNA-Extraction-of-Viruses-using-Wizard-Prep-Column-c26yhd" target="_blank">Cesium Chloride and DNA Extraction of Viruses using Wizard Prep Columns</a><br />4. <a href="https://www.protocols.io/view/Cesium-Chloride-Dialysis-for-Viruses-c7jzkm?guidelines" target="_blank">Cesium Chloride Dialysis for Viruses</a><a href="https://www.protocols.io/view/Cesium-Chloride-Dialysis-for-Viruses-c7jzkm?guidelines" target="_blank"><br /></a><br />Needed:<br />
<ul>
<li>Phage</li>
<li>Beckman Ti45</li>
<li>10% PEG</li>
<li>0.5M NaCl</li>
<li>Centrifuge 8K GSA</li>
<li>CsCl</li>
<li>Buffer</li>
<li>Tris</li>
<li>MgCl2</li>
<li>SW 28.1 Beckman Ultra Clear Tube</li>
<li>Needle</li>
<li>Syringe</li>
<li>Guage Needle</li>
<li>Stop cork grease</li>
<li>Plunger</li>
<li>Pierce Dialysizer (optional)</li>
<li>18 guage needle (necessary if using pierce dialysizer)</li>
<li>Tube</li>
<li>Mg buffer</li>
</ul>
To prepare a CsCl solution of a particular density, the percent by weight of CsCl can be calculated by the formula:<br />% wt/wt = 137.48 - 138.11/p<br />where 'p' is the desired density. For example, for p = 1.7 g/mL, use 56.24g CsCl and 43.76 mL H2O.<br /><br /><strong><strong>Grams of CsCl to be added to Buffer<br /><br /></strong></strong>
<table style="height: 223px;" width="472">
<tbody>
<tr>
<td>p</td>
<td>M</td>
<td>25 mls</td>
<td>50 mls</td>
<td>75 mls</td>
<td>100 mls</td>
<td>200 mls</td>
</tr>
<tr>
<td>1.3</td>
<td>2.4</td>
<td>10.10</td>
<td>20.20</td>
<td>30.31</td>
<td>40.41</td>
<td>80.82</td>
</tr>
<tr>
<td>1.4</td>
<td>3.2</td>
<td>13.47</td>
<td>26.94</td>
<td>44.16</td>
<td>53.86</td>
<td>107.76</td>
</tr>
<tr>
<td>1.45</td>
<td>3.6</td>
<td>&nbsp;</td>
<td>30.24</td>
<td>&nbsp;</td>
<td>60.48</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>1.5</td>
<td>4</td>
<td>16.87</td>
<td>33.74</td>
<td>50.61</td>
<td>67.48</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>1.55</td>
<td>4.4</td>
<td>18.48</td>
<td>36.96</td>
<td>&nbsp;</td>
<td>73.92</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>1.6</td>
<td>4.8</td>
<td>20.2</td>
<td>40.40</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>1.65</td>
<td>5.2</td>
<td>21.89</td>
<td>43.78</td>
<td>&nbsp;</td>
<td>87.55</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>1.7</td>
<td>5.6</td>
<td>23.57</td>
<td>47.15</td>
<td>70.7</td>
<td>94.29</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>
<br />(Yamamoto 1970 Virology 40, 734-744)

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Minimal growth medium for fission yeast. Very useful when performing transformations and needing to create dropout media. This protocol creates medium that yeast can grow on natively. To create dropout medium, simply omit whichever amino acid&nbsp; you are using as a marker.

[STEPS]
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TITLE: DNA extraction protocol for the eastern banjo frog using the Gentra Puregene Tissue Kit
AUTHORS: Qiye Li, Qunfei Guo, Yang Zhou, Huishuang Tan, Terry Bertozzi, Yuanzhen Zhu, Ji Li, Stephen Donnellan, Guojie Zhang

[DESCRIPTION]
Genomic DNA was extracted from the liver of an adult female Limnodynastes dumerilii dumerilii  using the Gentra Puregene Tissue Kit (QIAGEN, Hilden, Germany) with modifications as outlined in this protocol.

[STEPS]
3. .  
20 s 
5 min 

4.   
5.  
-20 °C 
120 min 

 


6.  
Room temperature 
60 min 
 


1. The following protocol is a modification of the protocol for "DNA purification from 5-10mg fresh or frozen solid tissue" using the Gentra Puregene Genomic DNA Purification Kit. 

The amounts listed in this protocol are for a single extraction. Four replicate extractions were made using this protocol.


Approximately 30mg of frozen liver tissue was minced with a scapel blade on ice.
2. 


 
55 °C 
  
100 °C 
10 min 
37 °C 
60 min 
Room temperature
TITLE: Fluorescent false neurotransmitter (FFN) live-cell DAT imaging
AUTHORS: gurvir.virdi

[DESCRIPTION]
This protocol describes how to measure the rate of uptake of FFN102, a fluorescent DAT substrate inside midbrain dopaminergic neurons. 

[STEPS]
1. To measure the presence and activity of the DAT, we utilised the commercially available fluorescent DAT and VMAT2 substrate FFN102 (Abcam, ab120866). 
2. To measure the uptake of the FFN102 dye, a field of view was first found using the brightfield settings on a Zeiss LSM 880 confocal microscope.
3. Cells were washed 1x with HBSS at room temperature
4. The cells then had 10 μM of the FFN102 dye in HBSS added and a time-series with an exposure every 5 seconds using the 405 nm laser was started, to measure uptake of the dye into the cells.
 

5. As a control to confirm specificity, samples in a different well were pre-treated with 5 μM of the DAT inhibitor nomifensine (Sigma) for 10 minutes in HBSS.

 

6. Once the dye had entered cells, the cells were depolarised by the addition of 50 mM KCl to observe FFN102 dynamics.
7. Rate of fluorescence intensity increase inside cells was measured across the time-series to give a readout for the rate of FFN102 uptake through the dopamine transporter.
TITLE: Stringency wash buffer
AUTHORS: Anna Schmidt, Sarah Nagel, Matthias Meyer

[DESCRIPTION]
Protocol for the preparation of Stringency wash buffer for automated single-stranded DNA library preparation using the ssDNA2.0 method (Gansauge et al. 2020).

References
Gansauge, M.-T., Aximu-Petri, A., Nagel, S., & Meyer, M. (2020). Manual and automated preparation of single-stranded DNA libraries for the sequencing of DNA from ancient biological remains and other sources of highly degraded DNA. Nature Protocols, 15, 2279-2300.

[STEPS]
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p><strong>Reagent List:</strong></p>
<ul>
<li>Sterile PBS</li>
<li>Cell culture medium (RPMI 1640 supplemented with 10% FBS)</li>
<li>Sterile plastic petri dishes</li>
<li>Sterile T-75 culture flask</li>
<li>RBC Lysis Buffer (Cat. No. 420301)</li>
<li>Concanavalin A (Con A) (Sigma, Cat. No. C5275)</li>
<li>Anti-mouse CD3ε, clone 145-2C11 (LEAF™ format, Cat. No. 100314)</li>
<li>Anti-mouse CD28, clone 37.51, (LEAF™ format, Cat. No. 102112)</li>
<li>Recombinant mouse IL-2 (carrier-free) (Cat. No. 575402)</li>
<li>Recombinant mouse IL-4 (carrier-free) (Cat. No. 574302)</li>
<li>Monensin Solution (Cat. No. 420701)</li>
</ul>

[STEPS]
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TITLE: MoClo reaction
AUTHORS: Laura Sánchez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Molecular cloning system </div></div>

[STEPS]
?. Fill in Setup Sheet (tab below)
?. Dilutions for Parts and Destination VectorsFollow instructions on "Dilutions_PRINT" sheetIf dilutions are already done, skip to "Reaction Set-up" below.
?. Reaction Set-up
?. Turn on PCR machineThaw DNA samplesThaw 10x NEB ligase buffer on ice
?. ___ uL of each part___ uL  destination vector___ μL sterile H2O0.5 μL of Promega T4 ligase enzyme
?. 
?. Obtain 1 PCR tube per reaction and labelAdd the following to each tube in order:2μL of NEB ligation buffer (10x)
?. 
?. Incubate as follows in PCR machine:            Level 1 or 2: 25 cycles of [37°C 1.5min, 16°C 3min], 50°C 5min, 80°C 10min, hold at 4°CUse in transformation or store at -20°C until use           Level 1 or 2: 25 cycles of [37°C 1.5min, 16°C 3min], 50°C 5min, 80°C 10min, hold at 4°C
TITLE: No Title
AUTHORS: 

[STEPS]
SECTION: Prepare the nucleic acid samples
?. 
SECTION: Prepare the nucleic acid samples
?. 
SECTION: Prepare the nucleic acid samples
?. 
SECTION: Assay design and chip ordering by Thermo Fisher
?. 
SECTION: Perform the Quant Studio™ 12K Flex system
?. 
SECTION: Perform the Quant Studio™ 12K Flex system
?. 
SECTION: Perform the Quant Studio™ 12K Flex system
?. 
SECTION: Perform the Quant Studio™ 12K Flex system
?. 
SECTION: Transfer and analyze experiment results
?. 
SECTION: Transfer and analyze experiment results
?. 
SECTION: Transfer and analyze experiment results
?. 
SECTION: Transfer and analyze experiment results
?.