eamag/protocols-with-steps huggingface dataset:
TITLE: Gradient PCR with DMSO 
AUTHORS: Ashwinuday  

[DESCRIPTION]
This protocol can be used to confirm genes or DNA of interest from a template using PCR. Also to amplify required amount of genes to larger amounts.

Gradient is used to find out the best annealing temperature. 

DMSO is required for the Pfu polymerase to work properly.

[STEPS]
1. Prepare the working stock solution of all the reagents required. If already prepared and stored, takeout from the refrigerator and thaw on ice.

2. Prepare the following mixes each of total 50 µL and 5 test samples were made for the 5 different annealing temperatures to be varied at each of the following percentages of DMSO used (0, 5, 10): -  

 


3. Perform PCR using a Thermo cycler with the following temperature settings (All temperatures are in degree Celcius).r 
In the thermo cycler gradient PCR option is kept and the following gradient is specified. Then the 5 test samples at each percentage of DMSO are labelled based on their temperature and kept in the correct column in the thermo cycler.

 
Set the 2nd, 3rd and 4th steps to repeat 30 times (or maximum till 35)
 

4. 
 

To prepare the 1% agarose gel, mix 0.5 g of agarose in 50 mL of 1X TAE buffer and heat well to form a clear solution. 


 

5. Then the mix is allowed to cool till it is bearable to touch. Carefully add 2 µL of EtBr to the mix and swirl well.

6. Then into a set gel cast with the appropriate comb (which is decided by the number of wells and the size of the wells required), pour the mix without bubbles. 
 

7. Once the gel solidifies, carefully take it out with a gloved hand and place in the buffer tank with enough buffer to cover the immersed gel and remove the comb without breaking the wells.
8. Mix approximately 10 µL samples with 1X loading dye and load into each gel. Load a 1kb ladder (or any suitable ladder) into the first lane. Run the gel at 100V

9. After the dye front crosses 75%of the gel length, stop the current.
10. Take the gel out (with gloved hands) by letting all the buffer out into the tank and then image the gel on UV.
Compare the sample bands with the ladder to estimate the bands of interest

11. Check bands corresponding to samples from which temperature shows good intensity and is at the right size and choose that annealing temperature for further PCRs
TITLE: Post-Surgical Dissection of Fallopian Tubes
AUTHORS: Stephen Fisher, Marielena Grijalva, Rong Guo, sarahjoh, Hieu Nguyen, John Renz, Jean G Rosario, Steven Rudich, Brian Gregory, Junhyong Kim, Kate O'Neill

[DESCRIPTION]
This protocol describes dissection of the Fallopian tubes in preparation for 10X Visium, 10X Multiomics, pathology review, and biobanking.  The Fallopian tubes have an inner mucosa surrounded by a muscular layer that is coated by serosa.  When sectioning the Fallopian tube the muscule layer may retract or protrude and distort orientation.  This can be addressed by using a stabilizing hand or dissection pins.

[BEFORE_START]
Dilute phosphate buffered saline (Gibco; 14200-075) to 1X with nuclease-free water.

[STEPS]
1. After removing uterus and Fallopian tubes from ice, dry each Fallopian tube and use marking dye to label the superior edge (12 o’clock), inferior edge (6 o’clock), anterior surface (3 o’clock) and posterior surface (9 o’clock).
2. Measure the Fallopian tube from cornua to fimbriated end.

  

3. Using a disposable scalpel, divide one Fallopian tube at the cornua and excise mesosalpinx.
4. Weigh Fallopian tube in a disposable weigh boat on ice.
5. Place on ice the uterus with the remaining contralateral Fallopian tube.
6. Remove the first Fallopian tube from ice and place on a cutting surface.
7. Divide Fallopian tube into four sections: isthmus, ampulla, infundibulum, and fimbriae.
8. Working one section at a time, slice tissue into four sections.
9. Using disposable weigh boats, weigh each tissue piece.
10. Tissue can be processed with protocols OCT-Embedded Tissue Preparation, Tissue Fixation Preparation, or Snap-Frozen Tissue Preparation depending on the desired downstream processing.
11. Remove the uterus with the remaining contralateral Fallopian tube from ice and repeat above steps.
TITLE: Simultaneous detection of miRNA and mRNA at the single-cell level in plant tissues
AUTHORS: Chi-Chih Wu

[DESCRIPTION]
Detecting the simultaneous presence of a microRNA (miRNA) and a mRNA in a specific tissue can provide support for the prediction that the miRNA regulates the mRNA. Ｗe develop a method that uses sequence-specific miRNA-locked nucleic acid (LNA) and mRNA-LNA probes. Moreover, it augments the detection signal by rolling circle amplification, achieving a high signal-noise ratio at the single-cell level. Dot signals are counted for determining the expression levels of mRNA and miRNA molecules in specific cells. We show a high sequence specificity of our miRNA-LNA probe, revealing that it can discriminate single-base mismatches. Numerical quantification by our method is tested in transgenic rice lines with different gene expression levels. 





[STEPS]
1. miRNA hybridization
 
      1. The slides with   sections are taken out the freezer and    equilibrated to RT for 40 mins.    2. Permeabilized in 20 ug/ml proteinase k   for proper duration    3. Quickly wash in DEPC-PBS    4. Quickly dehydrate the slides in EtOH   (50, 70, 99 %) and then air dry     5. Mount the secure seal reaction   chambers onto the slides.    6. Add 1x DEPC-PBS-tween 0.05 % (Wash   buffer) into the chambers to keep the slides wet until RT reaction ready  
 
3. mRNA cDNA synthesis
 
        Reagent  Stock  Final    NEB Tag DNA ligase  40U/ul  0.5 U/ul    Rnase H  5 U/ul  0.4 U/ul    Ribolock Rnase inhibitor  40 U/ul  1 U/ul    NEB Tag ligase buffer  10x  1x    BSA  20 ug/ul  0.2 ug/ul    KCl  1 M  0.05 M    Formamide  100%  20%    Pd_A  10 uM  0.1 uM    Pd_B  10 uM  0.1 uM    Pd_C  10 uM  0.1 uM      DEPC-H2O        
 
4. miRNA and mRNA padlock probe hybridization and ligation
 
        Reagent  Stock  Final    NEB Tag DNA ligase  40U/ul  0.5 U/ul    Rnase H  5 U/ul  0.4 U/ul    Ribolock Rnase inhibitor  40 U/ul  1 U/ul    NEB Tag ligase buffer  10x  1x    BSA  20 ug/ul  0.2 ug/ul    KCl  1 M  0.05 M    Formamide  100%  20%    Pd_A  10 uM  0.1 uM    Pd_B  10 uM  0.1 uM    Pd_C  10 uM  0.1 uM      DEPC-H2O        
 
5. rolling circle amplification
 
        Reagent  Stock  Final    Phi 29 polymerase  10 U/ul  1 U/ul    Ribolock Rnase inhibitor  40 U/ul  1 U/ul    10 X phi 29 buffer  10 x  1 x    dNTP  10 mM  0.25 mM    BSA  20 ug/ul  0.2 ug/ul    Glycerol  50%  5%      DEPC-H2O        
 
6. detection oligo hybridization
 
        Reagent  Stock  Final    Hyb mixture  4 x  2 x    L13535-S3-FITC  1 uM  0.1 uM    X01178-1852-Cy3  1 uM  0.1 uM    L11805-1854-Cy5  1 uM  0.1 uM      DEPC-H2O        
 
2. Section permeabilization
 
           Stock  Final    Formamide  100%  50%    SSC  20x%  5x    tRNA  10 mg/ml  0.5 ug/ul    Denhardt's  50x  1x    LNA probe A  10 uM  2-3 pmole    DEPC-H2O
TITLE: Managements for brain tumours cases during the COVID-19 pandemic. A protocol for  systematic review and meta-analysis.
AUTHORS: Mohammed A Azab, Sherief Ghozy, Abdulraheem Alomari, Ahmed Y Azzam

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Background and Objective:</span><span> The difficulties of neurosurgical brain tumours patients’</span></div><div class = "text-block">management and surgery during the COVID-19 pandemic are enormous and challenging </div><div class = "text-block">globally. the review will aim to review the managements have done with brain tumours cases </div><div class = "text-block">during the period of December 2019 to August 2020 amid the COVID-19 pandemic according to </div><div class = "text-block">the infection status of the patient and the status of the surgery.</div><div class = "text-block"><span style = "font-weight:bold;">Methods and Analysis:</span><span> This protocol follows the guidelines according to the Preferred </span></div><div class = "text-block">Reporting Items for Systematic reviews and Meta-Analysis Protocol (PRISMA) and registered </div><div class = "text-block">on the International Platform of Registered Systematic Review and Meta-analysis Protocols</div><div class = "text-block">(INPLASY). PubMed, Medline, Scopus and Google Scholar databases will be searched from </div><div class = "text-block">December 2019 to August 2020 for brain tumours, Infection status and status of the surgery. We </div><div class = "text-block">will perform study selection, data extraction and data analysis. The primary outcome of this </div><div class = "text-block">systematic review is to follow-up on the management lines that have been done with brain </div><div class = "text-block">tumours cases during the period of December 2019 to August 2020 amid the COVID-19 </div><div class = "text-block">pandemic according to the infection status of patients and status of surgery.</div><div class = "text-block"><span style = "font-weight:bold;">Ethical Approvals and Dissemination:</span><span> This systematic review study will not need ethical </span></div><div class = "text-block">approvals, because it doesn’t involve human beings. We aim to publish this systematic review in </div><div class = "text-block">a peer-reviewed journal. </div><div class = "text-block"><span style = "font-weight:bold;">Systematic Review Registration:</span><span> This protocol was registered with the International Platform </span></div><div class = "text-block">of Registered Systematic Review and Meta-Analysis Protocols (INPLASY) on 19 August 2020 </div><div class = "text-block">and was last updated on 19 August 2020 (registration number INPLASY202080082).</div></div>

[STEPS]
TITLE: Bacterial transformation
AUTHORS: Joris Van Asselberghs, Addgene The Nonprofit Plasmid Repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The following protocol is for bacterial transformation. To see the full abstract and additional resources, visit the </span><a href="https://www.addgene.org/protocols/bacterial-transformation?utm_source=abstract" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Addgene protocol page</span></a><span style = ":;">.</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/dpya7esn.jpg" /></div></div>

[STEPS]
?. Take competent cells out of  and thaw on ice (approximately - ).
-80 °C
?. Remove agar plates (containing the appropriate antibiotic) from storage at  and let warm up to room temperature and then (optional) incubate in  incubator.
4 °C
37 °C
?. Mix  -  of plasmid DNA (usually  - )    of competent cells in a microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few times.
1 µl
5 µl
10 pg
100 ng
1
*Pro-Tip*Transformation efficiencies will be approximately 10-fold lower for ligation of inserts to vectors than for an intact control plasmid.
?. Incubate the competent cell/DNA mixture on ice for -.
?. Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a  heat block for - ( is usually ideal, but this varies depending on the competent cells you are using).
42 °C
?. Put the tubes back on ice for .
?. Add - LB or SOC media (without antibiotic) to the bacteria and grow in  shaking incubator for -
250 µl
1000 µl
37 °C
*Pro-Tip*This outgrowth step allows the bacteria time to generate the antibiotic resistance proteins encoded in the plasmid backbone so that they will be able to grow once plated on the antibiotic containing agar plate. This step is not critical for Ampicillin resistance but is much more important for other antibiotic resistances.
?. Plate some or all of the transformation onto a 10 cm LB agar plate containing the appropriate antibiotic.
*Pro-Tip* We recommend that you plate   on one plate and the rest on a second plate. This gives the best chance of getting single colonies, while allowing you to recover all transformants.
?. Incubate plates up side down (agar side on top) at  overnight.
37 °C
*Pro-Tip*If the culture volume is too big, gently collect the cells by centrifugation and resuspend in a smaller volume of LB so that there isn't too much liquid media on the agar plates. If the agar plate doesn't dry adequately before the cells begin dividing, the bacteria diffuse through the liquid and won't grow in colonies.
?. clean bench with 70% ethanol
TITLE: Proteomics -Cell Lysis and Protein Digestion/Proteomics -Tandem Mass Tag Labeling 
AUTHORS: gustavo.parfitt

[DESCRIPTION]
Proteomics -Cell Lysis and Protein Digestion/Proteomics -Tandem Mass Tag Labeling

[STEPS]
1. For the proteomics your protocols were based on publish protocols with no modifications dx.doi.org/10.17504/protocols.io.bxa4pigw
TITLE: Agarose gel electrophoresis
AUTHORS: Brian P Teague

[DESCRIPTION]
We use agarose gel electrophoresis to analyze DNA samples -- is there DNA present? What size is it?

[BEFORE_START]
Before starting, make sure you have the DNA samples you'll be analyzing (either PCR or restriction digest) thawed on ice.

[GUIDELINES]
This protocol has been optimized so it can be run in a 2-hour laboratory section.

[STEPS]
SECTION: Cast the gel
1. Using a balance, weigh out 0.5 g in a weigh boat.

SECTION: Cast the gel
2. In a 250 Erlenmeyer flask, mix 50 mL, 0.5 g   , and 2 µL  DNA stain. Swirl to mix.
 

SECTION: Cast the gel
3. Microwave on HIGH for 30 s; the solution should begin to boil. Remove from the microwave and swirl.
 



SECTION: Cast the gel
4. Look carefully at the contents of the flask while you swirl it. If there are any flecks of undissolved agarose remaining, microwave again for 15 s. Repeat until the solution is completely clear.
 


SECTION: Cast the gel
5. Set the agarose solution on the bench to cool slightly. While it is cooling, insert the black wedges and the gel comb into the gel box. 
 

SECTION: Cast the gel
6. When the agarose solution is cool enough that you can hold your hand against it for 10 seconds, carefully pour the solution into the gel tray.
 

 

SECTION: Cast the gel
7. Wait 15 min for your gel to solidify.

 

SECTION: Cast the gel
8. Carefully remove the gel combs and the black wedges.

 

SECTION: Run the gel
9. If you removed your gel from the gel box (or cast it outside of the gel box), put your gel in the box with the wells toward the black electrode.


SECTION: Run the gel
10. Pour just enough 1X LAB buffer into the box to entirely submerge the gel.
SECTION: Run the gel
11. Below, select whether you're analyzing a PCR or a restriction digest:
SECTION: Run the gel
14. Carefully pipette 5 µL of    into the left-most well. Then, pipette your samples mixed with loading dye into successive wells.

SECTION: Run the gel
15. Assemble the top of the gel box onto the bottom. Plug the box into a power supply and run the gel at 10for 20 min.
 

 



SECTION: Run the gel
16. After you have run your gel, image it with a transilluminator.
SECTION: Run the gel
12. Carefully pipette 5 µL of    into the left-most well. Then, pipette your samples mixed with loading dye into successive wells.
 

SECTION: Run the gel
13. Assemble the top of the gel box onto the bottom. Plug the box into a power supply and run the gel at 10for 20 min.
 

 


SECTION: Run the gel
13. case 2    
SECTION: Run the gel
12. Cut a 1 cm x 4 cm (half a square) of   off of the roll.

SECTION: Run the gel
13. For each sample (not including the molecular weight standard), carefully deposit 1 µL of   onto the square of parafilm, then pipette 2 µL of the DNA sample onto the spot. Pipette up and down to mix.
 


SECTION: Run the gel
14. After you have run your gel, image it with a transilluminator.
TITLE: Efficacy, safety and complications of autologous fat grafting to the eyelids and periorbital area: A systematic review and meta-analysis
AUTHORS: Fan Yang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Background: In recent years, autologous fat grafting (AFG), also known as fat transfer or lipofilling, has been widely performed for periorbital rejuvenation and defect correction, although the evidence regarding its efficacy and safety is still lacking. Besides, with respect to the periorbital region, it is invariably the earliest appearance area of the facial aging phenomenon. Therefore, a systematic review and meta-analysis is needed to evaluate the efficacy and safety of this technique.</div><div class = "text-block">Methods: A literature search was performed in PubMed, Embase, and the Cochrane library databases on November 11, 2020, adhering to the PRISMA guidelines, to identify all relevant articles. Then, a data extraction and standardization process was performed to assess all outcome data. Ultimately, the data were assessed using a random effects regression model with comprehensive meta-analysis software. </div><div class = "text-block">Results: Thirty-six studies consisting of four cohorts and 32 case series with a total of 3997 cases were included. The mean follow-up was 1.5 years. Meta-analysis revealed a relatively high satisfaction rate of 93.7% (95% CI, 89.6%–96.2%). Frequent complications in 3997 patients receiving AFG were edema, chemosis, and contour irregularity, with an overall complication rate of 7.7% (95% CI, 4.3%–13.3%).</div><div class = "text-block">Conclusion: This systematic review and meta-analysis showed that AFG for rejuvenation of eyelids and periorbital area provided a high satisfaction rate and did not result in severe complications. Therefore, AFG might be performed safely for periorbital rejuvenation and reconstruction.</div></div>

[STEPS]
?. We will search the PubMed, Cochrane library, and Embase databases for articles published prior to November 11, 2020.
?. We will scrutinize the titles, abstracts, and full text of the retrieved articles and screen out articles that do not meet the inclusion and exclusion criteria.
?. We will perform  meta-analysis  by a comprehensive meta-analysis software (CMA), version 2.2.050 (Biostat, Englewood, NJ, USA). A random-effects  meta-analysis will be conducted to analyze all the evidence.
TITLE: DNA damage assessment in the adult Drosophila brain via comet assay
AUTHORS: Mel Feany

[DESCRIPTION]
This protocol describes how to determine DNA damage in the adult drosophila brain using the comet assay

[STEPS]
1. Prepare a Lysis solution for one slide by mixing 20 mL of lysis buffer (Trevigen Comet Assay Reagent Kit) and 2 mL of DMSO

2. Melt LMAgarose in a boiling water bath, aliquot to 1.5ml tubes, and place at 37 °Cuntil use

3. Place comet slide at 37 °Cuntil use

4. Dissect 2 adult fly brains per the desired genotype in ice-cold PBS
4.1. Homogenize brains with a blue pestle
5. Combine head homogenate with 100 µL 37 °C agarose and immediately pipette 75 µLonto Comet Slide

6. Place slides flat at 4 °C for 20 minutes
7. Immerse slides in prechilled Lysis Solution on ice for 45 min

8.  Immerse in a freshly prepared alkaline solution (prepare 25 mLl by mixing 0.3 g NaOH and 125 µL of 200mM EDTA in dH2O). pH>13 for 30 min at Room temperature 

9. Drain excess buffer and wash in 1X TAE twice for 5 min each

10. Run for 10 min at 23V/6mA in TAE_one volt per cm electrode to the electrode

11. Drain excess buffer and rinse in dH2O    

12. Immerse slides in 70% Ethanol for 5 min and then air-dry slides 5 min

13. The next day, prepare SYBR Green (Thermofisher) dilutant by mixing 1 µL in 10 mL of TE buffer (10 mM Tris-HCL pH 7.5, 1 mM EDTA in dH2O) and place 50 µL on each circle of the comet assay slide for 5 min 

14. Drain excess buffer and mount slides without DAPI in permount (Fisher Scientific)
15.  After mounting and drying the slides, take images with 20X objective of the epifluorescence microscope and analyze the comet tails using the Image J Comet assay plugin
TITLE: Cultivation protocol for anaerobic ciliates
AUTHORS: Johana Rotterova, Ivan Čepička

[STEPS]
?. [Sample isolation]
Cultivation protocol is followed by methods described in Rotterová et al. 2018 and Bourland et al. 2017.Isolate 10 ml of targeted sample from environments of interest (e.g., freshwater sulphidic sediment).
?. [Media preparation]
Prepare ATCC medium: 802 (Sonneborn's Paramecium medium) for freshwater ciliates or ATCC Medium: 1525 (Seawater Sonneborn's Paramecium Medium) for marine ciliates. Preparations for both below:
?. [Media preparation]
Instructions for 500 ml of ATCC #802 Freshwater Cereal Grass Media (Sonneborn's Paramecium medium) preparation:500 ml dH2O1.25 g cerophylle Boil for 5 minutesFilter through filter papersAdd 0.25 g Na2HPO4Autoclave at 121 °CAdd cerophyll to distilled water and boil for 5 minutes. Add 100 ml distilled water to compensate for evaporation. Filter through Whatman #1 filter paper and add 0.5 g Na2HPO4. Autoclave for 15 minutes at 121 °C.
?. [Media preparation]
Instructions for 1 liter of ATCC #1525 Seawater Cereal Grass Media (Seawater Sonneborn's Paramecium Medium) preparation:4 parts:Cereal Grass Medium:500ml bottle: Cerophylle500 ml dH2O2,5 g of cerophylleBoil for 5 minutes(it is twice as concentrated as the usual cereal grass media for freshwater cultures, + no Na2HPO4 included)Filter through filter papersArtificial seawater protocol:1 L bottle: SWA300 ml dH2O24.72 g NaCl0.67 g KCl1.36 g CaCl2.2H2O4.66 g MgCl2.6H2O100ml bottle: SWB100 ml dH2O6.29 g MgSO4.7H2O100ml bottle: SWC100 ml dH2O0.18 g NaHCO3Artificial seawater protocol:1 L bottle: SWA300 ml dH2O24.72 g NaCl0.67 g KCl1.36 g CaCl2.2H2O4.66 g MgCl2.6H2O100ml bottle: SWB100 ml dH2O6.29 g MgSO4.7H2O100ml bottle: SWC100 ml dH2O0.18 g NaHCO3100ml bottle: SWB100 ml dH2O6.29 g MgSO4.7H2O100ml bottle: SWC100 ml dH2O0.18 g NaHCO3100ml bottle: SWC100 ml dH2O0.18 g NaHCO3Autoclave separately at 121 °C, mix after cooling down.
?. [Cultivation establishment]
Add each 1 ml of the sediment four times into a 15 ml falcon tube with 10 ml of the designated media.
?. [Cultivation maintanance]
Reinoculate 1 ml of each sedimented culture into a new 15 ml falcon tube with 10 ml of the designated media every two weeks. Green organic matter from the cereal grass media will be gradually generated over time and the culture will lose anorganic particles, such as sand grains and soil, present in the original sample.If you have any questions, please contact me here or at johana.rotterova@natur.cuni.cz.