eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p> </p>
<p>The protocol described here is an adaptation from this paper:</p>
<p> </p>
<p>Sun, Z. Z., Hayes, C. A., Shin, J., Caschera, F., Murray, R. M., Noireaux, V. Protocols for Implementing an <em>Escherichia coli</em> Based TX-TL Cell-Free Expression System for Synthetic Biology. <em>J. Vis. Exp.</em> (79), e50762, doi:10.3791/50762 (2013).</p>
<p> </p>
<p>Please note that some steps were taken literally as they appear in that source, and It is highly recommended to see and study this paper before starting. </p>
<p> </p>
<p>Our adaptations include changes in the volumes of cell cultures and buffers, for smaller scale production. Also, it has been eliminated the clarification, ultra-centrifugation, and dialysis steps. Another change is the use of <em>E.coli</em> strain BL21 (DE3) STAR, instead of Rosetta 2 cells. All the above modifications allow the preparation of a simpler, and yet very effective cell extract suitable for cell-free RNAPT7 Tx-Tl reactions.  </p>
<p> </p>
<p> </p>
<p> </p>

[STEPS]
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TITLE: Tissue processing and freezing after surgery
AUTHORS: Bettina Ergün

[DESCRIPTION]
The aim of this protocol is to document the processing of fresh tissue after surgery.
 
The collected tissue must also be documented in the files which are linked down below
 
The protocol descibes in detail how to process the resulting tissue from following protocols:

Gewebesammlung Frischgewebe Zystektomie
Gewebesammlung Frischgewebe Prostatektomie
Gewebesammlung Frischgewebe Nephrektomie

in the current version.







[STEPS]
SECTION: Sample processing in the lab
1. Fresh tissue should be processed on the same day if possible, but can be stored at 4°C for a maximum of overnight.
To reduce pathogens, the tissue should be incubated for 60 min atRoom temperature 

SECTION: Sample processing in the lab
2.  
 

SECTION: Sample processing in the lab
3. 
 

SECTION: Sample processing in the lab
4. The tissue is divided in the lab into pieces with an edge length of 1-2 mm.
Mix the fragments of a tumor piece so that each aliquot is as representative as possible of the tumor area.
SECTION: Sample processing in the lab
5. 16 (decision from June 29, 2023; previously: 8) fragments are stored in 1 ml of freezing medium in a cryotube.
If there are enough fragments for more than 4 cryotubes, more fragments can be combined in one tube. A maximum of 5 cryotubes of each entity are frozen.
SECTION: Sample processing in the lab
7. Freeze samples in Mr. Frosty overnight.
SECTION: Documentation
9. Store patient data :
BIOBANKING und EVEs_ab 23052024.xlsx
SECTION: Documentation
10. Please add pictures of:
the orgin of the sample in the organ (if avaible)
each tissue piece that has been taken before cutting the fragments. The picture should include the tissue ID (in the picture)
the documentation sheet


 





SECTION: Sample processing in the lab
8. On the next day transfer the tubes to N2 in the right order and document the location in the N2 binder and the list mentioned in Step 9
SECTION: Sample processing in the lab
6. New procedure for storage and dokumentation started in May 2024 (change of storage tubes to LVL-Tubes with Barcode)
SECTION: Sample processing in the lab
6.1. scan tube numbers and fill in the list mentioned in Step 9

SECTION: Sample processing in the lab
6.2. Cryo tubes should be additionally labelled as follows:
Patient ID, Organ, T1/T2 resp. N1/N2...
No. of Pieces
Date
TITLE: Protocol 6: Selecting for Spizellomyces punctatus transformants
AUTHORS: Sarah M Prostak, Edgar M Medina, Erik Kalinka, Lillian Fritz-Laylin

[DESCRIPTION]
Once the co-culture IM plates have grown for four days, it is time to select Sp transformants. Selecting these transformants requires harvesting the cells from the co-culture plates, dislodging and removing any remaining Agro from the culture, and plating Sp onto K1 selection media. In our laboratory, we use 300 mg/L Hygromycin B (Gibco #10687010) as our selection marker. Additionally, we add 50 mg/L Carbenicillin (Carb) and 50 mg/l Tetracycline (Tet) to our K1 plates to kill Agrobacterium. All steps here, with the exception of centrifugation steps, must be carried out in a sterile environment, ideally in the sterile area around an open flame.

[STEPS]
SECTION: Steps
1. For each plasmid transformed, prepare a 50 mL conical with 30 mL of DS solution.

SECTION: Steps
2. From the appropriate conical for the plate to be harvested, remove 1 mL of DS.

SECTION: Steps
3. Distribute the DS among the four quadrants of the corresponding IM plate.
SECTION: Steps
4. Incubate at Room temperature for 5 min.

SECTION: Steps
5. Using forceps, sterilize both sides of a single edge razor blade with the flame.
SECTION: Steps
6. Tilt the rehydrated IM plate and gently scrape the surface of the agar and collect the growth into the DS pooled at the bottom.
SECTION: Steps
7. Rotate the plate and scrape along the agar until most of the opaque areas on the plate are gone.
SECTION: Steps
8. Use a 1000 μL pipette tip to scrap any remaining growth from the razor blade and resuspend the growth to the appropriate conical tube filled with DS. 
 

SECTION: Steps
9. Aspirate all of the liquid from the scraped plate and slowly dispense the liquid back into the appropriate conical tube. 
 

SECTION: Steps
10. From the top of the density gradient, remove 1 mL of DS.

SECTION: Steps
11. Wash the surface of the scraped IM plate several times with this fresh DS.
SECTION: Steps
12. Return the volume back to the conical.
SECTION: Steps
13. Invert the conical 3 times.
SECTION: Steps
14. Vortex the conical for 1-2 seconds to dislodge any remaining Agro from Sp cells.
SECTION: Steps
15. Repeat steps 2-14 for each plasmid transformed (i.e, for each IM plate you have)
SECTION: Steps
16. Centrifuge the conical(s) for 2000 rcf, 10 min.

SECTION: Steps
17. Check for the presence of a pinkish pellet, this is the chytrid Sp.
SECTION: Steps
18. Gently pour off the supernatant with the high side of the pellet facing up. 
 

SECTION: Steps
19. Gently resuspend the pellet into 500 µL of fresh DS.

SECTION: Steps
20. Pipette 200 µL of the resuspended Sp onto a K1 plate with selection antibiotics. 
 

SECTION: Steps
21. Spread the cells using 4-5 sterile glass beads.
SECTION: Steps
22. Once the plate is dry, remove the glass beads.
SECTION: Steps
23. Seal and incubate plates in a humidity chamber at Room temperature for 4 days or until colonies appear on the plate.
TITLE: cDNA synthesis using the Applied BiosystemsTM High-Capacity cDNA Reverse Transcription Kit
AUTHORS: Lisa-Maria Rosenthal, Giang Tong, Katharina Schmitt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">cDNA synthesis using the Applied Biosystems</span><span style = "font-weight:bold;vertical-align:super;">TM</span><span style = "font-weight:bold;">High-Capacity cDNA Reverse Transcription Kit</span></div></div>

[STEPS]
?. Allow the kit components to thaw on ice.Prepare the 2X RT master mix on ice.  AB1ComponentVolume (µL)210X RT Buffer2.0325X dNTP Mix (100 mM)0.8410X RT Random Primers2.05MultiScribeTMReverse Transcriptase1.06RNase Inhibitor1.07Nuclease-free H2O3.28Total per reaction10.0  Place the 2X RT master mix on ice and mix gently.
AB1ComponentVolume (µL)210X RT Buffer2.0325X dNTP Mix (100 mM)0.8410X RT Random Primers2.05MultiScribeTMReverse Transcriptase1.06RNase Inhibitor1.07Nuclease-free H2O3.28Total per reaction10.0
?. Pipette 10 μL of 2X RT master mix into individual tube.
?. Pipette 10 μL of RNA sample (800 - 1000ng total RNA) into each tube, pipetting up and down two times to mix.  Seal the plates or tubes.
?. Briefly centrifuge the tubes to spin down the contents and to eliminate any air bubbles.
?. Place the tubes on ice until you are ready to load the thermal cycler.
?. Program the thermal cycler using the conditions below.  ABCDE1SettingsStep 1Step 2Step 3Step 42Temperature25 °C37 °C85 °C4 °C3Time10 minutes120 minutes5 minutes∞
ABCDE1SettingsStep 1Step 2Step 3Step 42Temperature25 °C37 °C85 °C4 °C3Time10 minutes120 minutes5 minutes∞
TITLE: Analysis of the Chromosomal Localization of Yeast SMC Complexes by Chromatin Immunoprecipitation
AUTHORS: Vasso Makrantoni, Daniel Robertson, Adele L. Marston

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A plethora of biological processes like gene transcription, DNA replication, DNA recombination, and chromosome segregation are mediated through protein–DNA interactions. A powerful method for investigating proteins within a native chromatin environment in the cell is chromatin immunoprecipitation (ChIP). Combined with the recent technological advancement in next generation sequencing, the ChIP assay can map the exact binding sites of a protein of interest across the entire genome. Here we describe a-step-by step protocol for ChIP followed by library preparation for ChIP-seq from yeast cells.</div></div>

[STEPS]
TITLE: Detection and quantification of Candida spp. from subgingival areas
AUTHORS: Sanja Petrovic, Milena Radunovic, Ana Pucar

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Detection and quantification of Candida spp. from subgingival areas using two methods- sterile periodontal curette and sterile paper points. </div></div>

[STEPS]
?. 1. The first day- clinical periodontal examination (full mouth examination at six sites per tooth) in order to diagnose chronic periodontitis and detect the deepest periodontal pocket (with the highest PPD value).
?. -Record plaque index (Silness-Löe), bleeding on probing (BOP), periodontal pocket depth (PPD) and clinical attachment loss (CAL). Samples should not be obtained the first day, to avoid contamination by blood.
?. 2. The second day- sampling. Isolate sampling tooth with cotton rolls.
?. -Remove the supragingival plaque by a curette and sterile gauze.
?. -Place two sterile paper points (#30) into the pocket/sulcus until a mild resistance appeares and keep for 30 seconds. Paper points contaminated by blood should be excluded.
?. -Collect paper points and place into 1mL of Sabouraud dextrose broth.
?. - Use sterile curette to obtain samples of the complete subgingival biofilm and inoculated in separate sterile plastic tubes containing 1 mL of Sabouraud dextrose broth.-
?. - Send samples to microbiologic laboratory.
?. 3. The second day -microbiological protocols. Vortex samples for 60 seconds.
?. - Streak 20μl of suspended broth on SDA and ChromAgar using sterile plastic micro pipette.
?. -Incubate at 25°C for 48 hours
?. 4. The fourth day. Count the number of Colony Forming Units per sample.
?. -Multiply the obtained number by 50 in order to obtain CFU/ml.
TITLE: Enzyme–Ligand Interaction Monitored by Synchrotron Radiation Circular Dichroism
AUTHORS: Rohanah  Hussain, Charlotte S. Hughes, Giuliano  Siligardi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">CD spectroscopy is the essential tool to quickly ascertain in the far-UV region the global conformational changes, the secondary structure content, and protein folding and in the near-UV region the local tertiary structure changes probed by the local environment of the aromatic side chains, prosthetic groups (hemes, flavones, carotenoids), the dihedral angle of disulfide bonds, and the ligand chromophore moieties, the latter occurring as a result of protein–ligand binding interaction. Qualitative and quantitative investigations into ligand-binding interactions in both the far- and near-UV regions using CD spectroscopy provide unique and direct information whether induced conformational changes upon ligand binding occur and of what nature that are unattainable with other techniques such as fluorescence, ITC, SPR, and AUC.</div><br/><div class = "text-block">This chapter provides an overview of how to perform circular dichroism (CD) experiments, detailing methods, hints and tips for successful CD measurements. Descriptions of different experimental designs are discussed using CD to investigate ligand-binding interactions. This includes standard qualitative CD measurements conducted in both single-measurement mode and high-throughput 96-well plate mode, CD titrations, and UV protein denaturation assays with and without ligand.</div><br/><div class = "text-block">The highly collimated micro-beam available at B23 beamline for synchrotron radiation circular dichroism (SRCD) at Diamond Light Source (DLS) offers many advantages to benchtop instruments. The synchrotron light source is ten times brighter than a standard xenon arc light source of benchtop instruments. The small diameter of the synchrotron beam can be up to 160 times smaller than that of benchtop light beams; this has enabled the use of small aperture cuvette cells and flat capillary tubes reducing substantially the amount of volume sample to be investigated. Methods, hints and tips, and golden rules tomeasure good quality, artifact-free SRCD and CD data will be described in this chapter in particular for the study of protein–ligand interactions and protein photostability.</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A protocol for isolation of protoplast from plant cells adapted from Yoo et al. 2007 (http://www.nature.com/nprot/journal/v2/n7/full/nprot.2007.199.htm). Procedure was optimized for use on <em>Arabidopsis thaliana</em> tissue.</p>

[STEPS]
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TITLE: Sample prep - Stanford TMC
AUTHORS: John Hickey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>See our detailed protocol published with the following title: </span><span style = "font-weight:bold;">CODEX multiplexed tissue imaging with DNA-conjugated antibodies.</span></div></div>

[STEPS]
?. See our detailed protocol published with the following title: CODEX multiplexed tissue imaging with DNA-conjugated antibodies.
TITLE: Protein-A_Protein-LG Sandwich ELISA
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This ELISA was used to study the interactions between Staphylococcal protein-A (SpA) and protein-LA (SpLG) with different immunoglobulin preparations of mammalian and avian species.</div></div>

[STEPS]
?. Thisc ELISA was used to study the interactions between Staphylococcal protein-A (SpA) and protein-LG (SpLG) with different immunoglobulin preparations from mammalian and avian species. The 96 well microtiter plate was coated overnight at 4°C with 2 µg/µl per well of SpA in carbonate-bicarbonate buffer pH 9.6.
?. The plate was then treated with bovine serum albumin solution and washed 4X with PBS-Tween. 50 µl of immunoglobulins (1 mg/ml) is added and incubated for 1h at room temperature, and the microplate is rewashed 4X with PBS-Tween.
?. Then, 50 µL of o-phenylenediamine solution (4 mg/mL) was added, and the plate was incubated for 15 min at RT in the dark. The reaction was stopped with 50 µL of a 3M H2SO4 solution. xx
?. The plate was visually assessed for color development and read on a microplate reader at 492 nm. A cut-off point was calculated as the mean of the optical density of the negative controls multiplied by two. The cut-off point was set to 0.32.
TITLE: Epigenomic profiling of neuroblastoma cell lines
AUTHORS: Kristen Upton, Robyn T. Sussman, Khushbu Patel, Gregory P. Way, Rebecca N. Adams, Gregory I. Sacks, Rebecca N. Adams, Paolo Fortina, John M. Maris, Jo Lynne Rokita

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol explains the process of how we collected MYCN, MYC, and Histone ChIP-Seq data, as well as ATAC-Seq data for neuroblastoma cell lines. This protocol is comprised of three sections: Cell Growth and Expansion for care of neuroblastoma cell lines, ChIP-Seq protocol, and ATAC-Seq Protocol. Table 1, within the document, outlines which data was collected for each cell line. </div></div>

[STEPS]
?.
TITLE: Xylem inoculation with flg22 
AUTHORS: Cleo Bagchus

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Protocol can be used after: Plant Infection with </span><span style = "font-style:italic;">Xanthomonas Campestris </span><span>Campestris</span></div></div>

[STEPS]
?. [Day 1]
Place  of flg22 3 cm above the lesion on the central vein of the leaf.
15 µl
?. [Day 1]
With the needle puncture through the droplet into the central vein three times. Do not puncture the leaf completely. Due to a difference in pressure, the droplet should be sucked into the xylem.
?. [Day 1]
Place a second   of flg22 3.5 cm above the lesion on the central vein of the leaf.
15 µl
?. [Day 1]
Repeat step 2.
TITLE: Artificial Pith Gallery (APG) construction
AUTHORS: James Skelton, Jiri Hulcr

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to rear ambrosia beetles with the fungus of your choice. Could be used for just rearing beetles, or growing fungi on natural substrate. </div><div class = "text-block"><span>This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

[STEPS]
TITLE: TEST - manual DNA Purification via magnetic beads 
AUTHORS: Carla Jungkunz

[DESCRIPTION]
manual purification of DNA/PCRs via magnetic beads 

[BEFORE_START]
bring magnetic beads to room temperature (at least 30min)

[STEPS]
SECTION: prepare magnetic beads
1. 30 min bring magnetic beads to room temperature (RT)
SECTION: Binding of DNA
3. Add beads                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                            0.9 times the amount 
SECTION: 1st wash
7. add ethanol 
1.9 times the amount 
vortex thoroughly
SECTION: 2nd wash
10. add ethanol 
1.9 times the amount 
vortex thoroughly
SECTION: Airdry beads
13. 10 min keep lid open in magnetic rack
SECTION: Elute DNA
15. place tube in sample rack 
SECTION: prepare magnetic beads
2. vortex until homogeneous 
SECTION: Binding of DNA
6. remove supernatant
SECTION: Binding of DNA
5. 5 min place tube in magnetic rack 
SECTION: Binding of DNA
4. 5 min incubate for at least 5min
SECTION: 1st wash
9. remove supernatant
SECTION: 1st wash
8. 5 min place in magnetic rack 
SECTION: 2nd wash
12. remove supernatant
SECTION: 2nd wash
11. 5 min place in magnetic rack 
SECTION: Airdry beads
14. remove residual supernatant
SECTION: Elute DNA
18. transfer supernatant into labeled tube 
SECTION: Elute DNA
17. 5 min  place in magnetic rack 
SECTION: Elute DNA
16. add indicated volume of water and mix/vortex thoroughly
5 min incubate
TITLE: Plate Streaking
AUTHORS: Carlos Goller, Carly Sjogren

[DESCRIPTION]
Overview and Goals
Streaking bacteria on agar plates allows scientists to obtain isolated colonies of bacteria. An isolated colony provides up to 10^8 bacteria cells that are genetically identical. This is why we call them clones or refer to them as colonies. The medium we chose to streak out bacteria must provide the nutrients required for prolific organismal growth, and not all bacteria require the same nutrients. There are a variety of types of media and we will use Tryptic Soy Agar (TSA) plates to grow your bacteria, which may or may not support your bacterium's growth. 

After completing this lab you will gain the following lab skills:
Lab safety and proper personal protective equipment (PPE)
Proper use of equipment: bacterial cell culture incubator
Bacterial plate streaking
Bacterial plate incubation

[BEFORE_START]
Preparation
Learn how to perform bacterial plate streaking to obtain isolated colonies.
Watch Streaking for Isolation (3:38 min)
Read this article to learn about ways to streak a plate to obtain isolated colonies: Streak Plate Method- Principle, Types, Methods, Uses (15 min) 
Practice with a pen and paper the “Quadrant Streaking Method.” 
 
 

[STEPS]
SECTION: Procedure for plate streaking
1. Obtain a fresh petri dish containing TSA media.

SECTION: Procedure for plate streaking
2. Label the bottom of the plate (the side containing TSA media)  keeping to the circumference (write on the edge, not in the center) with your bacterial isolate#, your Team#, your initials and today's date. See the diagram below for a visual example. If the surface of the plate is wet, dry it off using a chemical wipe.

SECTION: Procedure for plate streaking
3. Obtain a sterile plastic loop.
SECTION: Procedure for plate streaking
4. Inspect the plate with the bacterial isolate you will characterize.
SECTION: Procedure for plate streaking
5. Using the sterile plastic loop, touch the loop to a single colony on your bacterial source plate.
SECTION: Procedure for plate streaking
6. Streak the bacteria on the loop onto your labeled inoculate plate following the inoculation procedure illustrated above. 
SECTION: Procedure for plate streaking
7. Put your lid back on your plate, turn it upside down (lid facing the benchtop) and bring it to the incubation chamber.
SECTION: Procedure for plate streaking
8. Incubate your bacterial plate overnight with the goal of obtaining single isolated colonies.
TITLE: U Cinn - Cholesterol Concentration
AUTHORS: Patrick Tso, Dana Lee

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:  </span></div><div class = "text-block">In vitro quantification of cholesterol in serum or plasma is determined using an Infinity Total Cholesterol Assay kit.  This assay enzymatically hydrolyzes the cholesterol esters in the sample to cholesterol and free fatty acids.  The free cholesterol is further oxidized and combined with HBA to allow it to be quantitatively measured in the serum or plasma. </div></div>

[STEPS]
?. Prepare working standards by making a serial dilution of the stock 200mg/dl standard.
?. Using a 96 well flat bottom plate, into separate wells, pipette 2μL of deionized water, standard, or sample to be assayed.
?. Add 200μL of Reagent (supplied ready to use) to all wells.
?. Incubate plate for 5 minutes at 37°C.
?. Determine the absorbance (abs) of the standards and of each unknown at 500nm.
?. Calculate values of unknowns from the standard curve.Specimen: Serum or Plasma. Specimen stable for 7 days at 2-8°C or 3 months at -20°C. Assay Linearity: 774 mg/dl Reagent Stability: Until Expiry on Bottle at 2-8°C Stability of Final Reaction: 60 minutes
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div class="page" title="Page 5">
<div class="section">
<div class="layoutArea">
<div class="column">
<p>The ISOLATE II Biofluids RNA Kit is specially developed for the rapid phenol-free isolation of high quality total RNA from biofluids and viruses. Total RNA can be purified from blood, plasma, serum and other types of biofluids such as saliva, urine, semen and cerebrospinal fluid (CSF). Viral RNA can be isolated from the same sample sources, as well as cultured cells and tissue. The kit isolates all sizes of RNA from large mRNA, viral RNA and ribosomal RNA (rRNA) down to small RNAs such as microRNA (miRNA) and short interfering RNA (siRNA).</p>
</div>
</div>
</div>
</div>

[STEPS]
?.