eamag/protocols-with-steps huggingface dataset:
TITLE: PCR normalization and size selection with magnetic beads
AUTHORS: Dominik Buchner

[DESCRIPTION]
This protocol describes how to clean up and normalize PCR products or DNA extracts and perform a size selection with carboxylated-magnetic beads and a PEG-NaCl buffer. It works by diluting the beads so that the binding capacity is lower than the PCR yield which leads to a normalization of all PCR products to the binding capacity. 

[BEFORE_START]
Make sure all buffers are prepared before starting.
For easier pipetting let the normalization solution adjust to Room temperature.

 


[GUIDELINES]
Follow general lab etiquette. Wear gloves to prevent contaminating the samples. Clean the workspace before starting with 80% EtOH. 

Ratio Guide:
To get an estimate the performance of different ratios the protocol was tested using a DNA Ladder  . The eluate was then measured using a Fragment Analyzer with the High Sensitivity Kit.

Input DNA:

 

Ratio 1.8:

 

Ratio 1:

 

Ratio 0.9:

 

Ratio 0.85:

 

Ratio 0.8:

 

Ratio 0.75:

 

Ratio 0.7:

 

Ratio 0.65:

 

Ratio 0.6:

 

Ratio 0.55:

 






[STEPS]
1. Shake the normalization solution until the beads are homogeneously resuspended
 

 

2. Add 31 µL  and 28 µL  to a 250 µL U-bottom assay plate

 

3. Add 9 µL 

4. To bind the DNA to the beads shake at   

 


5. Place the plate on a magnet to pellet the beads for 2 min 


 

 


6. Discard the supernatant by pipetting
7. With the plate still on the magnet, add 100 µL  to each sample

8. Incubate for at least 30 s


9. Discard the supernatant by pipetting
10.  

11. With the plate still on the magnet, incubate the plate for  5 min at Room temperature  to dry off residuals of wash buffer

12. Add 50 µL of elution buffer to each sample

13.   

14. Place the plate on a magnet to pellet the beads for 2 min

 


15. Transfer 40 µL of the DNA to a new PCR plate. Store at -20 °C
TITLE: Immunofluorescent staining for neuronal marker MAP2
AUTHORS: Qing Wang

[DESCRIPTION]
This is the protocol for immunofluorescent staining for neuronal marker MAP2.

[STEPS]
1. 1. Treat differentiated SH-SY5Y cells with 40 ug/mL eumelanin or pheomelanin or PBS for 24 hours.
2. 2. Process cells using Cytofix/Cytoperm™ fixation/permeabilization solution (BD554714, Thermo Fisher Scientific) and block with 5% normal goat serum.
3. 3. Add primary antibody against microtubule-associated protein 2 (MAP2)(30 µg/mL, OSM00036G, Thermo Fisher Scientific) and incubate at 4℃ overnight.
4. 4. Add secondary antibody (1:1000, Alexa 594-conjugated, A11012, Thermo Fisher Scientific) and incubate for 2 hours at room temperature.
5. 5. Stain nuclei with DAPI.
6. 6. For MAP2-positive cell counting, three images are captured randomly from each well using FluoView FV300 confocal microscope under a 60x objective lens. MAP2 and DAPI channels are merged, and MAP2-positive cells in each random visual field of 0.045000 mm2 are counted using ImageJ.
TITLE: MAGDM method based on some qROFDPPHM operators
AUTHORS: Yanru Zhong, Hong Gao, Xiuyan Guo, Yuchu Qin, Meifa Huang, Xiaonan Luo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><a href="" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">In this paper, a set of Dombi power partitioned Heronian mean operators of </span><span style = ":;font-style:italic;">q</span><span style = ":;font-style:italic;">-</span></a><span>rung orthopair fuzzy numbers (</span><span style = "font-style:italic;">q</span><span>ROFNs) are presented and a multiple attribute group decision making (MAGDM) method based on them is proposed. Firstly, the operational rules of </span><span style = "font-style:italic;">q</span><span>ROFNs based on the Dombi t-conorm and t-norm are introduced. According to these rules, a </span><span style = "font-style:italic;">q</span><span>-rung orthopair fuzzy Dombi partitioned Heronian Mean operator and its weighted form are then established. To reduce the negative effect of unreasonable </span><a href="" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">attribute</span></a><span> values on aggregation result in these operators, </span><a href="" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">a </span><span style = ":;font-style:italic;">q</span><span style = ":;">-rung orthopair fuzzy Dombi power partitioned Heronian mean operator and its weighted form are</span></a><span> constructed via combining them with the power average operator of </span><span style = "font-style:italic;">q</span><span>ROFNs. On the basis of the constructed operators, a method to solve the MAGDM problems based </span><span style = "font-style:italic;">q</span><span>ROFNs is designed. Finally, a practical example, a set of experiments, and comparisons are reported to demonstrate the feasibility and effectiveness of the proposed method. The demonstration results show that the method is feasible, effective, and flexible, which has satisfying expressiveness and concurrently can consider the interrelationships between different attributes comprehensively and reduce the negative influence of biased attribute values.</span></div></div>

[STEPS]
TITLE: Study design/methodology (Part 3 of &#34;Effects of Online Exercise Intervention on Physical and Mental Conditions in Young Adults with Chronic Neck Pain&#34;)
AUTHORS: Yiting Lin

[DESCRIPTION]
This is a Part 3 of "Effects of Online Exercise Intervention on Physical and Mental Conditions in Young Adults with Chronic Neck Pain".
Randomized clinical trial with 39 adults with self-reported chronic neck pain recruited. They were randomly assigned into two groups. The experimental group received online exercise therapy and the control group received conventional exercise therapy.Participants in both groups completed the same exercise program three times a week for six weeks with either face-to-face or online mode of delivery by physiotherapists.The degree of pain was assessed by the improvement in average and maximum VAS. Neck function and work limitations were assessed by NDI and WLQ respectively. HADS and its subscales were used to evaluate anxiety and depressive symptoms.

[GUIDELINES]
Study design/methodology:


Randomized clinical trial with 39 adults with self-reported chronic neck pain recruited. They were randomly assigned into two groups. The experimental group received online exercise therapy and the control group received conventional exercise therapy. 

Participants in both groups completed the same exercise program three times a week for six weeks with either face-to-face or online mode of delivery by physiotherapists. The degree of pain was assessed by the improvement in average and maximum VAS. Neck function and work limitations were assessed by NDI and WLQ respectively. HADS and its subscales were used to evaluate anxiety and depressive symptoms. Participants were assessed at baseline and at 6 weeks while the changes in WLQ were assessed biweekly. To ensure the effectiveness of online exercise therapy, therapists delivered the online exercise 3 times a week for 6 weeks, with videoconferencing once a week and posting of exercise record on WeChat app twice a week.

[STEPS]
TITLE: PBMCs isolation from CPT™ tube
AUTHORS:  Woong-Yang Park,  Jay Shin, Shyam Prabhakar

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the procedure for collection and isolation of blood samples using CPT tubes.</div></div>

[STEPS]
?. [Centrifugation and Collection of Blood Sample]
Centrifuge tube/blood sample at  in a horizontal rotor (swing-out head) for a minimum of  at  to .
0 Room temperature
Centrifuge: 1500 33
Centrifuge: 1800 33
Speed change of accel/decel: Soft
Remix the blood sample immediately prior to centrifugation by gently inverting the tube 8 to 10 times.
?. [Centrifugation and Collection of Blood Sample]
After centrifugation, mononuclear cells and platelets will be in a whitish layer just under the plasma layer.
?. [Centrifugation and Collection of Blood Sample]
Aspirate approximately half of the plasma without disturbing the cell layer.
?. [Centrifugation and Collection of Blood Sample]
Collect cell layer and transfer to a 50 mL size conical centrifuge tube with cap.
Collection of cells immediately following centrifugation will yield best results.
An alternative procedure for recovering the separated mononuclear cells is to re-suspend the cells into the plasma by inverting the unopened BD vacutainer® CPT™ Tube gently 5 to 10 times. This is the preferred method for storing or transporting the separated sample for up to 24 hours after centrifugation.
?. [Centrifugation and Collection of Blood Sample]
To collect the cells, open the BD Vacutainer® CPT™ Tube and pipette (use Pasteur Pipette) the entire contents of the tube above the gel into a separate tube.
?. [Cell Washing]
First Wash: Add wash buffer to bring volume to .
50 mL
?. [Cell Washing]
Cap tube and mix cells by inverting tube 5 times.
Avoid making bubbles if pipetting.
?. [Cell Washing]
Centrifuge at .
Centrifuge: 300 33, 15 min
?. [Cell Washing]
Aspirate as much supernatant as possible without disturbing cell pellet.
?. [Cell Washing]
Re-suspend cell pellet by gently vortexing or tapping tube with index finger.
?. [Cell Washing]
Second Wash: Add wash buffer to bring volume to .
20 mL
?. [Cell Washing]
Cap tube and mix cells by inverting tube 5 times.
?. [Cell Washing]
Re-suspend cell pellet in RPMI 1640 or wash buffer to bring to the concentration of 1×108cells/mL for counting.
?. [Cell Washing]
Take  for cell counting.
10 µl
?. [Cell Washing]
For cell counting, mix the cells with  (pipetting 10 times) and apply  of the mixture to a counting slide.
[Trypan Blue]
10 µl
?. [Cell Washing]
Count the cells using TCM automatic cell counter within  (the appropriate concentration: 5×104~1×107cells/mL).
?. [Cell Washing]
Centrifuge the remaining suspension at .
Centrifuge: 300 33, 10 min
?. [Cell Washing]
Aspirate as much supernatant as possible without disturbing cell pellet.
?. [Cell Washing]
Resuspend to freeze down in  in cryo tubes.
[of CryoStor CS10]
TITLE: Transfection of constructs in diplonemids to enhance the HR pathway, using RS-1 (3-(benzylamino) sulfonyl)-4-bromo-N-(4-bromophenyl) benzamide).
AUTHORS: Binnypreet Kaur, Drahomíra Faktorová , Julius Lukeš 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">RS-1 (RAD51-stimulatory compound 1) is a stimulator of homologous recombination (HR) protein RAD51 </div><div class = "text-block"><div class = "justify" style = "text-align:justify"> </div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div></div>

[STEPS]
?. Step 1: Countthe cells and plan to 1 nucleofection with 5x 107 cells for each construct.
?. Step 2: Harvestthe cells by centrifugation at 1300xg for 5 min at room temperature in Swing Bucket Rotor.
?. Step 3: Resuspendthe cell pellet in 100ul of AMAXA Human T- cell solution at 4C (from refrigerator combine 81.8ul of Human T-cell nucleofectorsolution + 18.2ul Supplement).
?. Step 4: Add 5-10ug of (PCR) (linearized DNA) along with 0.3 uM RS-1 enhancer into the cuvette (resuspend in 10ul of H2O).
?. Step 5: Put everything into the cuvette, close the cap and place in the electroporator, cuvette should only fit in one direction, but metal sides should face towards you.
?. Step6:  Press for the Program X-001 to electroporate and repeat the same as mentioned in tranformation protocol.
?.
TITLE: uDumBell – Circularization of rv0678 for genotypic bedaquiline resistance testing of Mycobacterium tuberculosis
AUTHORS: Jason D Limberis

[DESCRIPTION]
The ligation of dumbbell (hairpin) oligos to linear dsDNA produces pseudo-circular DNA. Including deoxyUridine in the PCR primer sequences causes Q5 and other high-fidelity polymerases to arrest elongation. This results in overhangs that were successfully ligated to a complementary hairpin structure. The deoxyUridine reduced the PCR product by approximately two-thirds, but this was ameliorated by increasing the Q5 DNA polymerase concentration three-fold.

[STEPS]
SECTION: Amplicon PCR
1. 
 

 
 
SECTION: Adapter ligation
2. Prepare the dumbell (hairpin) by incubating at 80 °C followed by cooling to room temperature over 30 min(this only needs to be done once)
SECTION: Exonuclease treatment – optional
5. 
 
Incubate at 37 °C for 30 min
Stop reaction by adding EDTA to at least 11 mM.
Heat Inactivation 70 °CC for 30 min

SECTION: Adapter ligation
3.  
 

Incubate as below, with the lid temperature set to 40 °C
 

SECTION: Adapter ligation
4. Incubate at Room temperature for 5 min
Place on amagnetic rack
Aspirate supernatant
Add 200 µL 70 % (v/v) ethanol
Wait for 30 s
Aspirate and discard the supernatant
Add 200 µL 70 % (v/v) ethanol
Wait for 30 s
Aspirate and discard the supernatant
Resuspend beads in 20 µL of H20 
Incubate for 2 min
Transfer to a clean PCR tube
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
The life cycle of phages can be characterized by one-step growth experiments, which are designed in a way that allows only a single infection cycle to take place (i.e., no re-infections occurring by phages produced during the experiment).

[BEFORE_START]
To limit the phage–host interaction in the experiment to a single infection cycle, phages and hosts have to be mixed in the right ratio. Prior to experiment, it is therefore necessary to determine the titer of the phage stock and to know the relation between cell density and optical density (i.e., obtain corresponding numbers of cells mL<sup>–1</sup> and OD) of the host. Infection should be done at low MOI (multiplicity of infection = ratio of phage to host) e.g., between 0.1 and 0.01. At higher MOI, the probability of cells infected by more than one phage would increase and the total estimate of infected cells becomes less than the phage input.

[GUIDELINES]
Originally developed by Ellis and DelbrÃ¼ck (1939), the one-step growth experiment measures the latent period and the burst size of a given phage on a given host (e.g., Adams 1959; Carlson 2005). Latent period and burst size are essential parameters in a description of phage properties and varies between phages and hosts and also with host growth conditions. The latent period is the minimum length of time it takes from adsorption of the phages to a host cell to lysis of the host with release of progeny viruses (Fig. 1). The burst size is the average number of phages released per infected host cell. The one-step experiment can be adapted to test the effects of different environmental factors on the infection process. For example, the burst size can be affected by the growth rate of the host (e.g., Middelboe 2000); they are expected to be higher when the host cell is nutrient replete and growing exponentially while one might expect a decrease in burst size if the host cells are under nutrient limitation.ÃÂ <br /><br />
<div style="text-align: left;"><img id="s-mce-img" class="s-mce-img" style="display: block; margin-left: auto; margin-right: auto;" src="https://s3.amazonaws.com/pr-journal/3jc2w6.jpg" alt="" data-src="https://s3.amazonaws.com/pr-journal/3ic2w6.jpg" data-ofn="Figure 1.jpg" /><br /><br /><strong>Figure 1:</strong>ÃÂ An example of the development in the number of plaque- forming units during a one-step growth experiment with a bacteriophage, and the definition of the parameters "Latent period" and "Burst size."ÃÂ <br /><br />One step growth experiments are often difficult to get to work for new phage host systems and adjustments to the standard procedure (e.g., the number of added phages, length of the experiment, sampling frequency, etc.) are often required depending on host growth rate, phage adsorption rate, infection efficiency, etc.<br /><br /></div>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Bicinchoninic acid Acid Protein Concentration measurement 
AUTHORS: JCPrice 

[STEPS]
?. Thaw the samples on ice.
?. Prepare standards using Pierce Protein Standard-2mg/ml, 1 mg/mL, 0.5 mg/mL,  0.25 mg/mL, 0.125 mg/mL, 0.062 mg/mL, 0.031 mg/mL,  0.016  mg/mL, 0  mg/mL
?. Organize your well plate and draw a map on the lid.  Group your standards and each dilution of your experimental standards to simplify the analysis.
?. Make dilutions depending on the amount of protein expected. For BioID samples, measure lysate and 1/5 dilution in lysis buffer, by loading 25ul of each sample dilution into 2 wells.  Label the lid to indicate where each sample and dilution is found
?. Load 25 ul of each standard into 2 wells.  Label the lid again.
?. Prepare the “Working Reagent” immediately before use by mixing CuSO4 and the Bicinchonic Acid Solution in a 1:50 ratio (need 200ul per sample or standard).
?. Add 200ul of Working Reagent into each well.  Use a multichannel pipet to quickly add the reagent to all wells containing sample or standard.
?. Begin the lag-time, a 15 min incubation of the microplate at 37oC with shaking
?. Read the absorbance at 562nm on plate reader (ProteinQuant_BCA)  C:/Program Files/Molecular Devices…/Protein Quant/BCA.ppr       (SoftMax Pro 5.2 rev C)  Analysis
?. Graph the standards with the absorbance on the x-axis and the concentration on the y-axis.  Standards with absorbance greater than 1.5 should not be used in the standard curve as the light signal is extremely low and overly noisy.
?. Fit standards data to a line using the trendline in excel or similar program.  The min and max absorbance represent the bounds for signal allowable for your unknown standards.  Although a polynomial fit could be used to fit the non-linear portions of a standard curve, this 9-point curve doesn’t adequately constrain higher order polynomials so it is better to reduce the range of the curve and focus on the linear region.
?. Using the parameters from the linear regression calculate a concentration for each unknown (x should be absorbance).  If multiple dilutions of the unknown are within the bounds of the standard curve these readings should be averaged together unless there is evidence that the measurement was done incorrectly i.e. replicates are extremely different.
TITLE: Non-cleavable Linkers
AUTHORS: BOC      Sciences

[DESCRIPTION]
Non-cleavable linkers require mAb degradation within lysosomes after ADC internalization to release the active drug. BOC Sciencesprovides synthetic non-cleavable linker products with a wide range of scales, purities, and delivery time options to meet your research needs. We ensure that each custom linker product is multiple-checked for quality via both mass spectrometry (MS) and high-performance liquid chromatography (HPLC) analyses during linker purification and quality control (QC) procedures after each step.

[STEPS]
TITLE: Diagnostic value of PD-L1 ≥ 1% expression status and its efficacy correlation with PD-1/PD-L1 inhibitor therapy in advanced solid cancers: a meta-analysis
AUTHORS: Anonymous

[DESCRIPTION]
Despite immune checkpoint inhibitors (ICIs) seem to have found their role in several hematological and solid tumors, several questions remain unanswered. Among these, the lack of validated biomarkers of response represents an important issue since only a proportion of patients benefit from immunotherapy. Based on these premises, a greater understanding of the role of potential biomarkers including programmed death ligand 1 (PD-L1) expression, tumor mutational burden (TMB), microsatellite instability (MSI) status, gut microbiota and several others is fundamental. In addition, clinical trials on immunotherapy widely differed in terms of drugs, patients, designs, terms of study phases, and inconsistent clinical outcomes.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Peptide purification counts after the synthesis, or other forms of making of it. For drug applied peptide like <a href="http://www.creative-peptides.com/services/cell-penetrating-peptides-synthesis-services.html" target="_blank">cell-penetrating peptides</a> are supposed to ba paid speical attention as impurities may bring unwanted effects, as well as APIs peptides like <a href="http://www.creative-peptides.com/product/bremelanotide-item-10-101-112-20.html" target="_blank">pt-141</a>.</p>
<p> </p>

[STEPS]
?. 
?. 
?. 
?.
TITLE: Electrophoretic mobility shift assay (EMSA)
AUTHORS: Olivier Boivin

[DESCRIPTION]
This protocol details an experimental procedure that can be followed to assess the DNA binding activity of a purified protein sample (recombinant yeast Cbf1 used here as example) to different sequences. DNA probes containing the desired sequences are formed by annealing complementary ssDNA oligos. These oligos are ordered from IDT and with 3' 6-FAM tags for imaging.

[STEPS]
SECTION: Probe annealing
1. Create storage stocks by resuspending ssDNA oligos to 100uM (100pmol/ul) in 1x IDTE buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0).
SECTION: Probe annealing
2. Create 10uM working stocks from the 100uM storage stocks. For 100uL of working stock:
 

SECTION: Probe annealing
3. Anneal labeled 1 units ssDNA probes to 1/1.2 units unlabeled probes in 100uL reactions. Using Thermocycler, heat reaction mixes to 95⁰C and incubate for ≥2min. Cool to 25⁰C/RT over 45min. Keep duplexed probes in the dark until they are needed.

 

SECTION: Protein binding reaction
4. Place running tank on ice and pre-run TBE native gel (8-20%; stored in backup -20⁰C)
at 200V for 60min or until current stabilizes.
SECTION: Protein binding reaction
5. Prepare 100uL of 5x protein (Cbf1) binding buffer:
 
 

SECTION: Protein binding reaction
6. Prepare individual binding reactions (20uL/reaction):
 
 

 

SECTION: Protein binding reaction
7. Incubate at RT in the dark for 50min following the addition of duplexed probe. 
SECTION: Protein binding reaction
14. 
14. 
SECTION: Electrophoresis
8. Add 4uL of orange G loading dye to each binding reaction (final volume = 24uL).
SECTION: Electrophoresis
9. Load 10uL of each binding reaction into well on pre-run TBE gel. Run at 200V for ~30min. Keep on ice and in the dark.
SECTION: Electrophoresis
10. Image gel using BioRad Chemidoc system in CIEMAS (same machine used for SDS-PAGE).