eamag/protocols-with-steps huggingface dataset:
TITLE: Oligo Pool Resuspension and PCR
AUTHORS: NUS iGEM

[DESCRIPTION]
2023 NUS-Singapore iGEM team followed this protocol to resuspend the dried oligo pool and perform PCR to amplify the DNA fragments in the oligo pool. 

[STEPS]
SECTION: Oligo Pool Resuspension
1. Centrifuge the oligo pool for a few seconds before opening the tube.
SECTION: Oligo Pool Resuspension
2. Calculate the volume of   required for the oligo pool (the final concentration of the sample must be at least 20ng/μL).

SECTION: Oligo Pool Resuspension
3. Add the respective volume of   into the tube with the oligos and resuspend the dried oligos by pipetting the solution.


SECTION: Oligo Pool PCR
4. Prepare a PCR tube, and add the following reagents: 

     KOD one      25μL        Each Primer      1.5μL        Template      1μL        DI Water      21μL   

SECTION: Oligo Pool PCR
5. Run a PCR with the following setting:

     98°C      10s        60°C      5s        68°C      10s        Go to step 1, repeat the cycle 10
  times        68°C      5 minutes        12°C      Infinite Loop   

SECTION: Oligo Pool PCR
6. Follow the PCR clean-up protocol to isolate the oligos from the PCR product and keep it at Room temperature .
TITLE: Effect of different cadences on a cycle ergometer on blood pressure in people with hypertension: an empty systematic review of randomized controlled trials
AUTHORS: Alexey Antonov, Alexander Miroshnikov, Polina Rybakova

[DESCRIPTION]
Current guidelines recommend exercise as part of primary and secondary prevention of CVD. However, little has been discussed about the effects of different cadences on a bicycle/exercise bike in the prevention and treatment of hypertension. Systematic responses directly related to exercise depend on the intensity, duration and frequency of exercise.
Based on the analysis of the problem situation, data from modern scientific periodicals and requests from sports physicians, clinicians and specialists in exercise therapy, the aim of the study was formulated.
The aim of the study is to systematically search for and summarize randomized controlled trials on the effect of different cadences on a bicycle ergometer on blood pressure in people with hypertension.

[BEFORE_START]
On October 1, 2024, we conducted a pilot screening according to the inclusion/exclusion criteria. We found that there was very limited data and conducted a full search strategy before protocol registration. We did not find any studies according to the inclusion/exclusion criteria. For this reason, this systematic review is designated as "empty".


[GUIDELINES]
The review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [Dickson]. The study protocol was written and registered before the search began and was not changed during or after the search, according to the Preferred Reporting Items for Systematic Review and Meta-Analyses Protocols (PRISMA-P) [Shamseer].
The literature search was carried out according to PRESS (Peer Review of Electronic Search Strategies) [McGowan].

[STEPS]
1.
TITLE: Protocols for Northern Analysis of Exosome Substrates and Other Noncoding RNAs
AUTHORS: Jonathan Houseley, Cristina Cruz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Over the past decade a plethora of noncoding RNAs (ncRNAs) have been identified, initiating an explosion in RNA research. Although RNA sequencing methods provide unsurpassed insights into ncRNA distribution and expression, detailed information on structure and processing are harder to extract from sequence data. In contrast, northern blotting methods provide uniquely detailed insights into complex RNA populations but are rarely employed outside specialist RNA research groups. Such techniques are generally considered difficult for nonspecialists, which is unfortunate as substantial technical advances in the past few decades have solved the major challenges. Here we present simple, reproducible and highly robust protocols for separating glyoxylated RNA on agarose gels and heat denatured RNA on polyacrylamide–urea gels using standard laboratory electrophoresis equipment. We also provide reliable transfer and hybridization protocols that do not require optimization for most applications. Together, these should allow any molecular biology lab to elucidate the structure and processing of ncRNAs of interest.</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><em>Sampling is for qPCR assay to determine total extracellular phage (qEXT), and for MPN (most probable number) assay to determine infective phage concentration</em>. <em>Samples containing phage and cells are filtered through a 0.2µm membrane in 96-well format to remove cells; filtrate fraction is saved for later analysis.</em></p>

[BEFORE_START]
<p><strong><em>Check the filter plates carefully for cracks and chips. We frequently receive plates that have been damaged in shipping. If the plate is cracked or damaged, you risk losing your samples in the centrifugation step.</em></strong></p>

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: Preparation of Agarose Gel
AUTHORS: NUS iGEM

[DESCRIPTION]
2023 NUS-Singapore iGEM team followed this protocol to prepare a 1% agarose gel. The team used this 1% agarose gel to isolate the DNA fragments from the common PCR and the colony PCR products. 

[STEPS]
1. For a 1% agarose gel, mix the following in a conical flask: 
5 g of Agarose powder.  
50 mL of 1x TAE buffer (Tris-Acetate-EDTA Buffer).   

2. Swirl the agarose solution to mix it well.
3. Heat the agarose solution in a microwave until it boils.
4. Take out the conical flask from the microwave and swirl the conical flask until the agarose solution is clear and without undissolved agarose powder or lumps.

 

5. Add 5 µL of Ultra GelRed gel stain into the agarose solution and swirl the conical flask until the colour becomes uniform.

6. Secure a gel tray tightly onto a gel caster and place an 8-well comb or 15-well comb into the tray.
7. Pour the agarose solution into the tray, ensuring that there are no bubbles.
8. Cool down the agarose solution for at least 30 min to get a solidified agarose gel.
TITLE: arc-melting
AUTHORS: Sterling G Baird

[DESCRIPTION]
Perform arc melting of various substances.

[BEFORE_START]
Measure and record the mass of each of the starting materials prior to arc melting.

[GUIDELINES]
The goal is to obtain a homogenous compound consisting of several constituent materials. The minimum total runtime required to obtain this homogeneous compound should be used which may require removing and observing the sample before placing it back into the arc melter for additional time.

[STEPS]
SECTION: Setup
2. 
  

 


SECTION: Setup
3. 
 
The argon gas tank should be set to   (right dial).

 

 

 
    

SECTION: Setup
4.   

SECTION: Setup
5.  

 
 

SECTION: Setup
6. 
 
Turn on the power supply.
SECTION: Arc Melting
7. 
 

 
Flip the power switch on for 5 s and then flip the power switch off.


 
If the materials haven't fully melted, run the arc melter for an additional 5 s  while moving the electrode across the top of the hearth using the shaft connected to the electrode. Only use the shaft above the arc melter to move the electrode.


  

 

SECTION: Disassemble
8. Wait 30 s  for the hearth to cool.

 
 
 
Remove the hearth from the arc melter.




 

If the compound consists of multiple individual pieces, use tweezers to place these back into the semi-spherical depression and remelt for 5 s (  ).

SECTION: Setup
1. Sum the masses of the individual components ( ).

 

SECTION: Calculations
9. Measure the weight of the final product, excluding the titanium (  ).

 

SECTION: Calculations
10. Subtract the final mass from the initial mass to determine the lost mass ( ).
TITLE: Sequential smFISH Allen Institute
AUTHORS: Jennie Close, Zoe Maltzer

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

[BEFORE_START]
Ensure all incubators and ovens are at the appropriate temperature prior to experiment. 

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

[STEPS]
SECTION: Tissue and Sectioning
1. 10-14 um cryosections are taken from fresh-frozen tissue, which are collected on poly-lysine-treated #1 coverslips at room temperature (RT). After 5-10 min at RT, sections are placed at 4°C until sectioning is complete. At that point, proceed immediately to fixation and permeabilization.
SECTION: Fixation/Permeabilization
2. Post-fix sections for 15 min with 4% PFA @ 4C
SECTION: Fixation/Permeabilization
3. Wash with PBS 3X
SECTION: Fixation/Permeabilization
4. Permeabilize with room temperature isopropanol 3  min
SECTION: Fixation/Permeabilization
5. Air dry for 30 min in fume hood (Stopping point: store coverslips at -80C)
SECTION: Fixation/Permeabilization
6. Optional: Treat sections with 8% SDS/PBS for 10 minutes, followed by 3 – 5 rinses with PBSor 2XSSC
7. Add 2ml 2X SSC
 
SECTION: Hybridization
8. Place sections in hyb  buffer without probes for 5 min.
SECTION: Hybridization
9. Add probes to  400ul hyb buffer at a final concentration of 2ng/ul* (specific to 6-well plate format – if using perfusion chamber, this volume can be reduced)

*We store a working 200ng/ul stock of probes in the dark at 4C. These are diluted 1:100 for hybridizations, but this may need to be adjusted depending on the probe.
SECTION: Hybridization
10. Incubate at 37 C for 2H
SECTION: Wash
11. Add 2 ml wash buffer to each well, incubate at 37 C for 15 min
SECTION: Wash
12. Remove wash buffer
SECTION: Wash
13. Add 2 ml fresh wash buffer and incubate at 37 C for 15 min
SECTION: Wash
14. Replace wash buffer with fresh wash buffer + DAPI (final 5ug/mL) and incubate at 37 C for 15 min
SECTION: Wash
15. (GLOX buffer step if performing antibody stain)
SECTION: Wash
16. Mount and image or store at 4 C in 2XSSC until imaging session
SECTION: Stripping
17. 65% formamide/2X SSC, 10 min  X 3, 30 C
SECTION: Stripping
18. 3 washes in 2XSSC
SECTION: Imaging
19. Add enzymes to Imaging Buffer just prior to imaging.
SECTION: Imaging
20.
TITLE: Squishing insects for preservation of HMW DNA in the field 
AUTHORS: Fiona Teltscher, Mara Lawniczak

[DESCRIPTION]
Ideal preservation for maximum recovery of high molecular weight (HMW) DNA from insects is to snap freeze living insects on dry ice and store at -70C. However,  dry ice or liquid nitrogen for snap freezing is not always accessible in the field. When snap freezing and ultra cold storage is not an option, gently compromising the cuticle by squishing the insect can improve the penetration of any preservation solution and thereby increase both the quantity and the quality of recovered DNA. Here we present a step by step protocol for how to carry out the squish procedure for mosquitoes. While we use mosquitoes as an example, this protocol should improve HMW DNA recovery for most adult arthropods when used in conjunction with an appropriate preservative such as 100% ethanol in excess, EDTA, or DMSO salt solution (DESS). It is advised to move room temperature squished insects in excess preservative to ultra cold storage as soon as possible. If the excess preservative will freeze solid (like DESS), and there is no further risk to cold chain before processing for HMW DNA extraction, we advise to remove the preservative solution before freezing. In our experience, we are able to retrieve good quality HMW DNA from single mosquitoes held in excess 100% ethanol or DESS at room temperature for 1 week and sequence this DNA using PacBio HiFi. Mosquitoes stored this way also resulted in good quality Hi-C data. 

[BEFORE_START]
Ensure all surfaces have been cleaned with 70-80% Ethanol (and ideally bleach before that). Have cleaning wipes for forceps.

[STEPS]
1. Transfer your live caught mosquito into    to kill it. 



2. 
With clean forceps remove the mosquito from   and gently dab it on a clean wipe (e.g. Kimtech wipe) to dry off any remaining ethanol. Transfer it into a 0.5 mL Eppendorf tube (or similar) filled with 400 µL of preservation liquid of your choice. 

[If you are using ethanol for preservation, you can skip this step and preserve directly in the ethanol that you killed the specimen in. Ensure there is sufficient ethanol in the tube to completely submerge the mosquito and not so much that you spill ethanol when you insert a pestle.]
 
 
 

 



3. With a clean pestle appropriate for the size of tubes that you are using and insects you are preserving, gently press the insect into the liquid and against the side of the tube until you feel the cuticle break. This ensures that the preservation solution can penetrate the tissue and increase the quality and quantity of DNA later extracted.
           
 

 


4. The mosquito is now ready to be shipped at room temperature or stored. In our experience, it is best to keep specimens at the coldest available temperature at all times so once you have access to a -70C freezer, remove the liquid (unless it is ethanol, which won't freeze) and snap freeze the specimen. This will make it easier to prepare the specimen for extraction as you won't have to wait for the preservative to thaw, potentially risking the quality of the DNA.
TITLE: ARDRA: amplified rDNA restriction analysis
AUTHORS: Eva Petrova, Roey Angel

[DESCRIPTION]
ARDRA: amplified rDNA restriction analysis

  1. Sklarz MY, Angel R, Gillor O, Soares MIM. Amplified rDNA Restriction Analysis (ARDRA) for Identification and Phylogenetic Placement of 16S-rDNA Clones. In: de Bruijn FJ, editor. Handbook of  Molecular Microbial Ecology I: Metagenomics and Complementary Approaches [Internet]. Hoboken, New Jersey: Wiley-Blackwell; 2011. p. 59–66. Available from: http://eu.wiley.com/WileyCDA/WileyTitle/productCd-0470644796,descCd-tableOfContents.html

  

[STEPS]
SECTION: PCR
1. A direct colony PCR is best used for this purpose, reaction mix might vary according to the specific Taq polymerase used. This following mix yields a strong amplification so that only 12 µL reaction is required. 
The following mix was used with Hy Labs' Taq polymerase. If different Taq is used, re-optimization might be required.
 
 
 
DNA template is added by poking a colony with a sterile tip and mixing with the mixture (don't use wooden toothpicks as they will absorb the liquid).
SECTION: PCR
2. 3-4 µL are loaded on agarose gel (1%) to verify products which should leave 5-8 µL of PCR product (because of various losses to evaporation and pipette errors). In this way the restriction can be done in the same tube of the amplification thus saving time and materials.
3 1
SECTION: Restriction
3. We use TaqI for the restriction though other enzymes could be use as well (see references). The same enzyme must be used throughout the scanning of the library for the purpose of creating a database. 5-10 µL of the PCR reaction (depending on amplification results) was mixed with 20U of enzyme and the appropriate amounts of buffer and water as described by the manufacturer. 
* Note that too much DNA per enzyme will result in incomplete digestion and an unresolvable pattern.
5 1
0 1
0 1
0 1
SECTION: Restriction
4. Tubes were incubated in a thermocycler overnight.
SECTION: Gel preparation running conditions
5. Gels are prepared with 3% agarose, we use low-melting (though this might not be necessary) in TBE 1X. Our gel frames are 25X15 and 10X15 cm respectively, for the large frame we use 250 mL TBE + 7.5 g of agarose and for the small frame we use 100 mL TBE + 3 g of agarose. Let to solidify for 30 min on the bench and 30 min at 4°C. 
SECTION: Gel preparation running conditions
6. The small gel is run on 70V for 3 hours and the large one on 90V for 5 hours. 
SECTION: Gel preparation running conditions
7. Following electrophoresis gels are post-stained for 20 min. in TBE 1X (the same buffer from the electrophoresis can be used) with 10 µL of EB (10 mg/mL) for each 100 mL of buffer and visualized under UV.
TITLE: Green Lab Nanoparticle Prep For In Vivo
AUTHORS: Edwin Yoo

[DESCRIPTION]
draft

[STEPS]
SECTION: Background (Materials in bold)
1. This protocol is to generate 3x100ul DNA nanoparticle injections at 40 w/w (DNA:polymer) and 0.25 mg/ml DNA.
-100 ul injections per mouse, 350 ul NP+DNA per frozen tube (thaw before animal injection).
-DNA stock should be 1 ug/ul
-Polymer stock should be 100 ug/ul
-For 40 w/w, 88 ug DNA/tube, 3500 ug polymertube
-Excipient is 90 ug/ul sucrose (stock sucrose is 500 ug/ul)
-Final volume 350ul before freezing (287 ul NP, 63 ul excipient)
-Sodium acetate pH 5
SECTION: Background (Materials in bold)
2. 
SECTION: Formulation Add In Sequence By Row
3.
TITLE: Reto F1016B
AUTHORS: Camila Miranda Bajo Gutiérrez, Diana Paulina Arroyo Soltero

[DESCRIPTION]
Pasos de como realizar el circuito que se necesita para el reto F1016B

[STEPS]
SECTION: Preparación del circuito
1. Reúne todos los materiales que se necesitan y coloca la protoboard en tu área de trabajo.
SECTION: Preparación del circuito
5. Conecte la sonda del osciloscopio y conectela a la protoboard.
SECTION: Ejecución del experimento.
6. Encienda la fuente de carga y calibre el voltaje a 5 Volts.
SECTION: Ejecución del experimento.
7. Encienda el osciloscopio 
SECTION: Ejecución del experimento.
8. Obtenga los datos
SECTION: Preparación del circuito
2. Visualiza el siguiente circuito
SECTION: Preparación del circuito
3.   

SECTION: Preparación del circuito
4. Utiliza los materiales para realizar la simulación del circuito en el protoboard.
TITLE: Flow Cytometry-Based Gamma-H2AX Assay for Radiation Biodosimetry
AUTHORS: Ghazi Alsbeih, Krishna Mishra, Maha Alrashd, Subramanian M. Pulicat, Najla Al-Harbi, Sara Bin Judia, Belal Moftah

[STEPS]
?. [A. Lymphocytes separation and fixation:]
Collect blood samples in heparinized Vacutainers (BD, USA).
?. [A. Lymphocytes separation and fixation:]
Irradiate 2 ml aliquot of whole blood with the required radiation dose.
?. [A. Lymphocytes separation and fixation:]
Incubate for 30 minutes (or other time) at 37ºC.
?. [A. Lymphocytes separation and fixation:]
Take the 2 ml whole blood and dilute (1:1) with 2ml PBS in a 15 ml tube.
?. [A. Lymphocytes separation and fixation:]
Carefully layer the mix on 2 ml of Histopaque (Ficoll).
?. [A. Lymphocytes separation and fixation:]
Centrifuge at 1,200 g (Eppendorf 5810 R) for 15 min at RT.
?. [A. Lymphocytes separation and fixation:]
Isolate middle cloudy lymphocytes layer and wash 2-3 times with cold PBS*.
?. [B. γH2AX immunostaining and flow cytometry measurement]
Wash fixedlymphocytes twice with PBS*.
?. [B. γH2AX immunostaining and flow cytometry measurement]
Incubate lymphocytesfor 1 hour at 37ºC with primary antibodies (anti-phospho-histone H2A.X, Ser139, clone JBW301; Millipore, CA, USA), in a 1:200 dilution in a buffercontaining 1% bovine serum albumin (BSA) and 0.12 % TX-100.
?. [B. γH2AX immunostaining and flow cytometry measurement]
Wash twice with PBS*.
?. [B. γH2AX immunostaining and flow cytometry measurement]
Incubate withsecondary antibodies (FITC-goat antimouse Ig BD Pharmingen, San Jose, CA, USA),in a 1:200 dilution in a buffer containing 1% bovine serum albumin (BSA) and 12 % TX-100, for 1 hour at 37◦C.
?. [B. γH2AX immunostaining and flow cytometry measurement]
Wash twice with PBS*.
?. [B. γH2AX immunostaining and flow cytometry measurement]
Suspend lymphocytes in fresh 400-500 μl of PBS according to cells concentration.
?. [B. γH2AX immunostaining and flow cytometry measurement]
Measure gamma-H2AXfluorescence in lymphocytes using flow cytometer (LSR II, Becton Dickinson, USA) and analyze the data using PC-basedBD FACSDiva software. The following settings are used:
?. [A. Lymphocytes separation and fixation:]
Fix lymphocytes(1-5x106) with 2% paraformaldehyde (PFA) for 10-20 minutes on ice. Atthis stage, fixed lymphocytes can either be processed further forimmunostaining or stored overnight at +4ºC.
?. [B. γH2AX immunostaining and flow cytometry measurement]
FITC laser excitationof 488 nm and emission of 533 nm.
?. [B. γH2AX immunostaining and flow cytometry measurement]
A single populationof lymphocytes is gated and used for mean fluorescence intensity (MFI) calculations.
?. [B. γH2AX immunostaining and flow cytometry measurement]
Yields of γH2AX arecalculated as the ratio of mean fluorescence intensity of irradiated versuscontrol samples.
?. [*Note: All PBS washing step include suspending cells in 500 μl PBS and centrifuging (Eppendorf 5810 R) at 100rpm for 10 minutes.]
TITLE: Measuring mitophagy via FACS with mtKeima reporter
AUTHORS: Louise Uoselis

[DESCRIPTION]
Preparation of samples for measuring mitophagy levels using mtKeima reporter by FACS

[STEPS]
SECTION: Day 1
1. Seed cells in a 24 well plate, aiming for a confluency of ~80-90% at the time of treatment. Seed additional wells of cells not expressing any fluorescent proteins, cells expressing only mtKeima, and cells expressing only YFP-Parkin (to be used as gating controls).
SECTION: Day 2
2. Feed all cells with standard growth media for 60 min prior to treatment 

SECTION: Day 2
3. Replace media in each well with media containing the drug you are treating with.
NOTE: Do not change the media or treat the additional wells of cells to be used for gating control.
SECTION: Day 2
4. 60 min prior to harvesting, feed the untreated wells with 0.5 mLof fresh growth media. 

SECTION: Day 2
5. At the conclusion of the treatment timepoint, harvest the cells using the following procedure:
SECTION: Day 2
5.1. Aspirate media from all wells
SECTION: Day 2
5.2. Wash all wells once with 500 µLof room temperature PBS

SECTION: Day 2
5.3. Add 150 µLof trypsin to each well, and incubate cells at 37 deg C for 90 s

SECTION: Day 2
5.4. Place plates onto ice, and harvest each sample into a separate microfuge tube on ice by resuspending each sample with 500 µL of ice cold standard growth media

SECTION: Day 2
6. Centrifuge all samples at 1000x rcf for 90 s at 4 deg C

SECTION: Day 2
7. Carefully aspirate the supernatant from all samples
SECTION: Day 2
8. Resuspend each sample in 50 µL of FACS media and place into FACS analysis tubes. Keep samples on ice until immediately prior to analysis.