eamag/protocols-with-steps huggingface dataset:
TITLE: 1.2 Bradford assay
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protein concentration estimation using Bradford assay</div></div>

[STEPS]
?. [Protein concentration estimation using Bradford assay]
Prepare working reagent by diluting 1part Dye Reagent Concentrate with 4 parts distilled, deionized water. This diluted reagent may be used for approximately 2 weeks when kept at room temperature.
?. [Protein concentration estimation using Bradford assay]
Prepare dilutions of a protein standard (Get from Becker lab- Sarah, they have aliquots for the diluted standards). The linear range of the assay for BSA is 0.2 to 0.9 mg/ml
?. [Protein concentration estimation using Bradford assay]
Add 50ul of each standard into the cuvettes and add 2.5 ml of Bradford reagent (optimized from 100 ul + 5 ml)
?. [Protein concentration estimation using Bradford assay]
Dilute your samples with 0.1% SDS in the cuvettes. For 1:10 dilution add 45ul of 0.1% SDS to the cuvette and add 5 ul of your sample (40 ul of 0.1% SDS and 10 ul sample for 1:5), mix properly. Add 2.5 mL of Bradford reagent to each tube and mix.
?. [Protein concentration estimation using Bradford assay]
Incubate at room temperature for 5 minutes. Absorbance will increase over time; samples should incubate at room temperature for no more than 1 hour.
?. [Protein concentration estimation using Bradford assay]
Measure absorbance at 595 nm.
?. [Protein concentration estimation using Bradford assay]
Plot the standard curve using standards and measure OD and results for the samples. Multiply the result with the dilution factor to get the concentration of your protein sample.
?. [Protein concentration estimation using Bradford assay]
Use an excel sheet and calculate the amount of you sample to be mixed with the appropriate sample buffer to get a final concentration of 0.5 ug/ul.
TITLE: Anthropometric knee study in patients with Osteoarthritis: Evaluation of possible differences between genders
AUTHORS: Fabrício Bolpato Loures, Rogério Franco de Araújo Góes, Eduardo Branco de Sousa, Naasson Cavanellas, João Maurício Barretto, Marcel Jun Sugawara Tamaoki, Rodrigo Sattaminni Pires e Albuquerque, Pedro José Labronici

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The data collection protocol was used for research involving the anthropometric study of the knee in patients with osteoarthritis during total knee arthroplasty (TKA) surgery. These data allowed comparing the dimensions of the knees with the available implants, investigating the existence of possible variations between the sexes and correlating the measurements with quantitative and qualitative data for each individual. In addition, it made it possible to define the profile of patients undergoing TKA in our services.</div></div>

[STEPS]
TITLE: Lab 5--Cloning a PCR Fragment into a Plasmid (Paper Activity)
AUTHORS: Harley King

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This activity includes a pdf containing instructions for creating a paper plasmid and PCR fragment (gene of interest). I remember doing this with undergraduates with another professor but I couldn't find a similiar activity online. So I recreated it. </div><div class = "text-block">This paper activity helps students with:</div><div class = "text-block">1) scanning and finding restriction sites</div><div class = "text-block">2) contemplating sticky end vs blunt digestion</div><div class = "text-block">3) visualizing cloning concepts using tactile materials</div></div>

[STEPS]
?. [Download and Print Activity]
Download and print the "
?. [Create PCR Insert]
Use scissors to cut out the three lines of “PCR Insert." Overlap the lines and secure them with tape. Keep the PCR Insert in a line. Do not loop.
?. [Create the Plasmid]
Use scissors to cut out the seven lines of “Vector Backbone." Overlap the lines and secure them with tape. Clear off your bench, this is about 6-ft long!Connect the beginning of line 1 to the end of line 7 to form a loop. This represents a circularized vector or plasmid.
?. [Annotate PCR Insert]
Beginning on the left side of the PCR Insert, scan for a start codon. Underline it. Continue scanning for a stop codon. Box it. Use a marker or highlighter to color the bases between the start and stop codon.
?. [Annotate PCR Insert]
Translate the codons into amino acids. Write the one-letter-abbreviation underneath the coding strand.
?. [Annotate PCR Insert]
Scan upstream and downstream of the PCR Insert for palindromic sequences representing endonuclease target sequences e.g. GAATTC, EcoRI. Outline the target sequences and write the name of the endonuclease that recognizes the sequence. Identify the cleavage sites within the sequence.
?. [Find Restriction Sequences in the Plasmid]
Beginning anywhere in the vector backbone loop, scan for palindromic sites representing endonuclease cleavagesites. Outline the cleavage sites and write the name of the endonuclease that recognizes the site.
?. [Digest and Ligate]
Pick a 5’ cleavage site in the PCR Insert. Using scissors, cut the double stranded DNA the same way the endonuclease would cleave.Repeat with a 3’ cleavage site in the PCR Insert.
?. [Digest and Ligate]
Locate the same 5’ PCR Insert cleavage site in the “Vector Backbone” and cut.Locate the 3’ PCR Insert cleavage site in the “Vector Backbone”. Cut.
?. [Digest and Ligate]
Tape the digested PCR Insert into the digested “Vector Backbone”. Verify that the overlaps are complimentary
TITLE: Crosslinking Immunoprecipitation Beads
AUTHORS: Bryon Drown, Caroline DeHart, Kelleher Research Group

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Immunoprecipitation with non-covalent capture of antibodies is a rapid and effective method for enriching samples with desired antigens. Interference by the antibody in down-stream analysis of intact proteins by mass spectrometry can be eliminated by covalent linkage of antibodies to magnetic beads. While several amine-reactive bifunctional linkers have been used for this application, dimethyl pimelimidate (DMP) are particularly advantageous due to its water-solubility, stability of resulting amidines at low pH, and retention of positive charge at reaction sites. By cross-linking to Protein A/G magnetic beads, a greater portion of antibodies are correctly oriented and able to engage their antigen. Cross-linking antibodies to Protein A/G beads with DMP is a quick and effective method that mitigates interference from heavy and light chain IgG in down-stream mass spectrometry applications.</div></div>

[STEPS]
?. [Complex Formation]
Form complex with beads and antibody
?. [Complex Formation]
Add 900 uL Binding Buffer and 100 uL magnetic beads to 1.5 mL LoBind tube.
?. [Complex Formation]
Collect beads with magnet and resuspend with 900 uL Binding Buffer.
?. [Complex Formation]
Add 50 ug IgG antibody.
?. [Complex Formation]
Incubate overnight with rotation.
4 °C
?. [Crosslink Beads]
Prepare fresh triethanolamine and ethanolamine buffers. DMP should be added to Crosslinking Buffer immediately before use.
?. [Crosslink Beads]
Collect beads with magnet and remove supernatant. Wash beads with 1 mL TEA Wash Buffer.
?. [Crosslink Beads]
Resuspend beads with 1 mL Crosslinking Buffer and incubate at ambient temperature for 1 hr while rotating.
0 Room temperature
?. [Crosslink Beads]
Collect beads with magnet and immediately wash beads with 1 mL Quenching Buffer. Resuspend beads in 1 mL Quenching Buffer and incubate at ambient temperature for 15 min while rotating.
0 Room temperature
?. [Crosslink Beads]
Collect beads with magnet and wash beads with 1 mL TEA Wash Buffer, 1 mL Basic Elution Buffer, 1 mL Acidic Elution Buffer, and 1 mL TBS twice.
?. [Crosslink Beads]
Store beads in 1 mL Storage Buffer at
4 °C
TITLE: Bioinformatic procedure for shotgun metagenomic analysis of human saliva microbiota
AUTHORS: Victoria Meslier, Florence Thirion , Florian Plaza-Onate , Emmanuelle Le Chatelier , mathieu.almeida

[DESCRIPTION]
This protocol describes the bioinformatical procedure for the study of human saliva microbiota.

[BEFORE_START]
NA

[GUIDELINES]
NA

[STEPS]
SECTION: General description
1. Read Quality check
SECTION: General description
2. Mapping and microbial gene count procedure
SECTION: General description
3. Metagenomic Pangenome Species MSP profiles
TITLE: UC Davis - Uninary Albumin Excretion (UAE) Protocol
AUTHORS: Peter Havel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">
Albumin blue dye is a stain for the specific and sensitive spectrofluorometric determination of albumin in natural matrices. AB 580 binds to the albumin present in urine samples and the fluorescence can be quantified using a fluorimeter.</div></div>

[STEPS]
?. Prepare working reagent.
?. Prepare standards by serially diluting 200 mg/l standard 1:1 to make 100, 50, 25, 12.5, 6.25 standards.
?. Add 25 μl of standard and sample to each well.
?. Add 125 μl of working reagent. Read in fluorimeter using 590 nm excitation and 616 nm emission.IMPORTANT: Make sure not to add any bubbles to the wells when dispensing reagents, this will interfere with reading in the platereader.
?. Use a polynomial 2nd order curve fit to construct a standard curve. Interpolate the values of the unknowns using the standard curve.
TITLE: Bold SW 1 NV modified medium
AUTHORS: Roscoff Culture Collection

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Medium used for some eukaryotes</div><div class = "text-block">Adapted from CCAP web site :  https://www.ccap.ac.uk/media/documents/BB.pdf</div><div class = "text-block">See also : http://www.ife.ac.uk/ccap/MediaRecipes.html#bb</div></div>

[STEPS]
?. Change from standard Bold medium  ABCDE1Volume
for 500 ml
Component
Concentration Stock Solutions
25 ml
CaCl2
CaCl2.2H2O
0.5 gr
200 ml
35 ml
NaNO3
NaNO3
16 gr
200 ml
45 ml
MgSO4
MgSO4.7H2O
10 gr
200 ml
510 ml
PO4
From PCRS11 media
65 ml
NH4Cl (K)
From K media
&nbsp;
&nbsp;
73 ml
K trace metal
From K media
&nbsp;
&nbsp;
850 µl
Vitamines
&nbsp;
&nbsp;
&nbsp;
9467 ml
SW sterile
&nbsp;
&nbsp;
&nbsp;
.
ABCDE1Volume
for 500 ml
Component
Concentration Stock Solutions
25 ml
CaCl2
CaCl2.2H2O
0.5 gr
200 ml
35 ml
NaNO3
NaNO3
16 gr
200 ml
45 ml
MgSO4
MgSO4.7H2O
10 gr
200 ml
510 ml
PO4
From PCRS11 media
65 ml
NH4Cl (K)
From K media
&nbsp;
&nbsp;
73 ml
K trace metal
From K media
&nbsp;
&nbsp;
850 µl
Vitamines
&nbsp;
&nbsp;
&nbsp;
9467 ml
SW sterile
&nbsp;
&nbsp;
&nbsp;
TITLE: RNA Isolation from Plant Tissue Protocol 15: Hot Acid Phenol Method for Algae
AUTHORS: 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Implemented by: Falicia Goh and Neil Clarke</div><div class = "text-block"><span>This RNA isolation method is modified from that described by Köhrer and Domdey</span><span style = "vertical-align:super;">5</span><span>.</span></div><div class = "text-block"><span>This protocol is part of a collection of eighteen protocols used to isolate total RNA from plant tissue. (RNA Isolation from Plant Tissue Collection: </span><a href="https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6</span></a><span>) </span></div><div class = "text-block"><span style = "vertical-align:super;">5 </span><span>Kohrer, K. & Domdey, H. Preparation of high molecular weight RNA. Methods in Enzymology 194, 398‐405 (1991).</span></div></div>

[STEPS]
?. Preheat phenol and phenol:chloroform to . Heated phenol should not be re-used.
65 °C
?. Collect algae cells via centrifugation for  at  at .
0 Room temperature
?. Re-suspend frozen pellet in  of preheated extraction buffer.
800 µl
?. Immediately add  of hot acid phenol.
800 µl
?. Incubate at  for . Vortex every 1 min for .
65 °C
?. Centrifuge at  at  for .
4 °C
?. The aqueous phase was transferred to fresh 1.5 ml micro-centrifuge tube.
?. Repeat steps 5 - 7. Repeat 3x (depending on the amount of cells used).
?. Extract with equal volume of phenol:chloroform (5:1).
?. Transfer aqueous phase to a new 1.5 ml microfuge tube. Volume should be ~.
700 µl
?. Add 1/10 volume of   sodium acetate, pH 5.5, and 1 volume of isopropanol.
?. Spin in micro-centrifuge at  at top speed for .
4 °C
?. Remove the supernatant without dislodging the pellet.
?. Wash the pellet with 70 % ethanol.
?. Invert and air dry tubes at room temperature.
?. Flash freeze pellets with liquid nitrogen and keep at  until extractions are carried out.
-80 °C
?. Vortex the tubes for .
?. Vortex for  at .
0 Room temperature
?. Spin for  in a microcentrifuge at top speed.
?. Repeat step 9 three times.
?. Hold at  for  or more.
4 °C
?. The pellet was re-suspended in  DEPC treated H2O.
50 µl
?. The RNAs were stored at .
-20 °C
TITLE: OMS Atlas FFPE Spatial Mapping
AUTHORS: Brett   Johnson, Danielle Galipeau, George Thomas

[DESCRIPTION]
This protocol describes the procedure by which the OMS Atlas serially sections a FFPE block, prepares the resulting slides, and then distributes the specimens for downstream analysis.

[BEFORE_START]
Transfer FFPE blocks to OHSU Knight Histopathology Shared Resource (HSR) for sectioning and processing. Coordinate execution of this sectioning protocol to allow for shipping within the same business week.

[STEPS]
SECTION: Preparation
1. Verify the identity of the FFPE block to be cut against written request for sectioning.
SECTION: Preparation
2. Label all slides with a unique BEMS ID and slide number, corresponding to the written request and FFPE spatial map (below).
 

SECTION: Sectioning
3.  Align block on microtome to minimize tissue loss.
SECTION: Sectioning
4. Face into block at 5μm until full section of tissue is achieved.
SECTION: Sectioning
5. Cut adequate ribbon at 5μm to cover all 8 serial sections.
SECTION: Sectioning
6.  Mount tissue sections onto appropriate slide types, maintaining serial order and orientation of sections.
SECTION: Processing
7.  Perform hematoxylin and eosin (H&E) staining on slides #1 and #8.
8. Bake slides #2, 3, 5, and 6.

8.1. Place slides #2, 3, 5, and 6 in 55 °C oven overnight.
8.2.  The next morning, raise the oven temperature to 65 °C for up to 60 min.
Note: Slides should be baked at 65 °C for at least 30 minutes.

8.3.  Remove slides from oven. Place slides in slide boxes and store at room temperature.
9. Deliver slides #2 through #7 to BioLibrary for distribution.
TITLE: Hematologic alterations and early mortality in a cohort of HIV positive African patients
AUTHORS: Fausto Ciccacci, Francesca Lucaroni, Roberto Latagliata, Laura Morciano, Elisa Mondlane, Moises Balama, Dyna Tembo, Jane Gondwe, Stefano Orlando, Leonardo Palombi, Maria Cristina Marazzi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify">Infection with Human Immunodeficiency Virus (HIV) is highly prevalent worldwide, especially in Sub-Saharan Africa, where anaemia is also widespread. HIV infection is known to be associated with anaemia and various other haematologic alterations, but little data on correlation with immunological and virologic conditions in treatment-naïve patients is available. To investigate these associations, we conducted a retrospective analysis of baseline data (general details, nutritional status, full blood count and HIV infection progress data) and 12 months follow-up status for HIV+ adult patients in 22 health facilities in Malawi and Mozambique.</div><style>
							  .justify:after {
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							    display:inline-block;
							    width: 100%;
							  }
							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">Among the 22.657 patients included, we found associations of sex, nutritional status, CD4 count, and VL with anaemia, leukopenia, and thrombocytopenia. Also, any cytopenia was present in 1/3 of patients with normal nutritional status and less advanced HIV infection, and it wouldn’t be diagnosed in a basic HIV care setting.</div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">Moreover anaemia, lower Red blood cells and platelets counts correlated with mortality in the first year of care, independently by BMI, Hb, CD4 count and VL.</div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div><div class = "text-block"><div class = "justify" style = "text-align:justify">Our results emphasize the need for including a full blood count in the routine HIV care services in Sub-Saharan Africa.</div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
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							</style></div></div>

[STEPS]
?.
TITLE: General methanogens: mcrA mlas-mod  – mcrA-rev
AUTHORS: Eva Petrova, Roey Angel

[DESCRIPTION]
Amplification of the marker gene methyl coenzyme M reductase alpha subunit (mcrA) using the general primers mlas-mod – mcrA-rev
 
mlas-mod    GGY GGT GTM GGD TTC ACM CAR TA    Angel et al. (2011), Plos One
mcrA-rev      CGT TCA TBG CGT AGT TVG GRT AGT   Steinberg and Regan (2008), AEM.
 
Fragment size: 472bp.

[STEPS]
SECTION: PCR mixture
1.  
  
  
  

SECTION: PCR program
2. 1.  94◦C – 5′
2.  x 5 {
       a.  60◦C – 1°C - 45''
       b. 72◦C – 30''
       c.  94◦C – 30'
      }
 3.  x 29 {
        a.  55◦C – 45''
        b.  72◦C – 30''
        c.  94◦C – 30''
       }
 
4.  55◦C – 45''
5.  72°C - 10'
TITLE: TH DAB staining
AUTHORS: Pranay  Srivastava, Waijiao Cai, Xiqun Chen

[DESCRIPTION]
This protocol details the stating procedure for TH DAB staining.

[STEPS]
SECTION: TH DAB staining
1. Wash a full set of sections (6-8 sections) in a well in PBS, 5 min × 3.
SECTION: TH DAB staining
2. Incubate sections in 1% H2O2 (1 mL 30% H2O2 in 29 mL TBST), 30 min at Room temperature.

SECTION: TH DAB staining
3. Wash sections in TBST 5 min × 3.
SECTION: TH DAB staining
4. Block sections in 5% NGS/TBST, 60 min at Room temperature.

SECTION: TH DAB staining
5. Incubate sections in rabbit anti-TH (#SA-497), 1:3000 in TBST, 4 °C 60 min.

SECTION: TH DAB staining
6. Wash sections in TBST, 10 min × 3.
SECTION: TH DAB staining
7. Incubate sections in anti-rabbit biotinylated (#OS03B), 1:3000, 60 min at 4 Room temperature.

SECTION: TH DAB staining
8. Prepare ABC (Avidin-Biotin-Complex, Vectastain ABC Kit, # PK-6100): 1 drop A and 1 drop B in 2.5 mL TBST, at least 30 min before use.

SECTION: TH DAB staining
9. Wash sections in TBST, 10 min × 3.
SECTION: TH DAB staining
10. Incubate sections in ABC, 30 min at 4 Room temperature.

SECTION: TH DAB staining
11. Prepare DAB solution (Sigma FAST, DAB Peroxidase Substrate Tablet Set), 1 DAB and 1 Urea Hydogen Peroxide tablet in 15 mL distilled water.

SECTION: TH DAB staining
12. Wash sections in TBST, 10 min × 3.
SECTION: TH DAB staining
13. Incubate sections in 1 mL DAB, 1 min-2 min.

SECTION: TH DAB staining
14. Wash sections in TBST, 5 min × 3.
SECTION: TH DAB staining
15. Mount the sections on positive charged plus slides.
SECTION: TH DAB staining
1.1. Wash a full set of sections (6-8 sections) in a well in PBS, 5 min (wash 1/3) (if stored in cryoprotective solution).

SECTION: TH DAB staining
1.2. Wash a full set of sections (6-8 sections) in a well in PBS, 5 min (wash 2/3) (if stored in cryoprotective solution).
SECTION: TH DAB staining
1.3. Wash a full set of sections (6-8 sections) in a well in PBS, 5 min (wash 3/3) (if stored in cryoprotective solution).
SECTION: TH DAB staining
3.1. Wash sections in TBST  5 min (wash 1/3).
SECTION: TH DAB staining
3.3. Wash sections in TBST  5 min (wash 3/3).
SECTION: TH DAB staining
3.2. Wash sections in TBST  5 min (wash 2/3).
SECTION: TH DAB staining
6.1. Wash sections in TBST, 10 min (wash 1/3).

SECTION: TH DAB staining
6.3. Wash sections in TBST, 10 min (wash 3/3).

SECTION: TH DAB staining
6.2. Wash sections in TBST, 10 min (wash 2/3).

SECTION: TH DAB staining
9.1. Wash sections in TBST, 10 min (wash 1/3).

SECTION: TH DAB staining
9.2. Wash sections in TBST, 10 min (wash 2/3).
SECTION: TH DAB staining
9.3. Wash sections in TBST, 10 min (wash 3/3).
SECTION: TH DAB staining
12.1. Wash sections in TBST, 10 min (wash 1/3).

SECTION: TH DAB staining
12.3. Wash sections in TBST, 10 min (wash 3/3).
SECTION: TH DAB staining
12.2. Wash sections in TBST, 10 min (wash 2/3).
SECTION: TH DAB staining
14.1. Wash sections in TBST, 5 min (wash 1/3).

SECTION: TH DAB staining
14.3. Wash sections in TBST, 5 min (wash 3/3).
SECTION: TH DAB staining
14.2. Wash sections in TBST, 5 min (wash 2/3).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is the procedure used to cluster MAGs for the <em>Tara Oceans</em> dataset assembled by Benjamin Tully and Elaina Graham</p>

[BEFORE_START]
<p>Before starting be sure you have downloaded Binsanity. Please see the <a href="https://github.com/edgraham/BinSanity" target="_blank">Binsanity github page</a> for full installation instructions and a walkthrough of parameters for each program.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Immunofluorescent staining of formalin-fixed OCT-embedded human lung tissue
AUTHORS: Justinn  Barr, Jamie   Verheyden, Xin  Sun

[DESCRIPTION]
This protocol describes steps for immunofluorescent staining of formalin-fixed paraffin-embedded human lung tissue.

[STEPS]
1. 2X5 min wash in Phosphate-buffered saline with Tween (PBSTW) in a slide holder on a flat rotator.
2. Blocking:
2.1. Mix up 5% serum (50 µL  serum / 1 mL of PBSTW)
 



2.2. Remove old PBSTW from the sample and add block.
2.3. Cover with Hybrislip.
2.4. Let sit in a humidified chamber at Room temperature for ≥ 1 hour.

3. Primary antibody:
3.1. Mix up more of the 5% serum.
3.2. Add antibody to the serum at the desired concentration. 
3.3. Take the humidified chamber to a cold room.
3.4. Add primary antibody in the cold room.
3.5. Cover with Hybrislip.
3.6. Seal the humidified chamber.
4. On Day 2:

Wash slides 3X10 min at Room temperature  in slide holder on a flat rotator. 

5. Secondary antibody:
5.1. Mix up 5% serum.
5.2. Add secondary antibody to the serum at the desired concentration.
5.3. Add antibody mix to the slides.
5.4. Let it sit in a humidified chamber EITHER at Room temperature  for 1 hour OR in the cold room overnight.
5.5. Cover with Hybrislip.
6. Wash:
6.1. 1x10 min in PBSTW with DAPI.
6.2. 2x10 min in PBSTW.
7. Mount the slides in VectaShield and seal with nail polish.
TITLE: Feeding 6wp
AUTHORS: Andrea Argouarch

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for feeding dural cells after outgrowth from tissue in a 6 well plate. </div></div>

[STEPS]
?. [Observations]
Record observations Biopsy pieces should look dense and dark; it will not change morphologyClean and straight edges on the tissue is best Outgrowth will vary between a few days to 2 weeks, depending on biopsyAfter a week, gently change media and then feed every 2-3 days        o Be careful not to move coverslip and to not tilt plate
?. [Preparation]
Turn off UV lights and clean hood with 70% ethanol
?. [Preparation]
Clean items with 70% ethanol and bring into hood    a. Sterile Filtered Media with PenStrep
?. [Culturing]
Carefully remove old media with vacuum line, making sure not to touch the coverslip.
?. [Culturing]
Do not tilt plate while feeding to prevent coverslip from sliding
?. [Culturing]
Add 3 mls   of new media per well, making sure not to touch the coverslip    a. Add media slowly    b. Make sure the coverslip is still firmly and evenly over the biopsy
3 mL
?. [Culturing]
Gently place 6 well plate in incubator (5% CO2, 37oC)
37 °C
?. [Culturing]
Continue to gently feed every 2 – 3 days for 3 weeks to allow enough cells to emerge and expand from tissue
?. [Clean Up]
Throw away biohazard materials properly
?. [Clean Up]
Clean and sterilize hood with 70% ethanol and turn on UV
?. [Clean Up]
Update cell culture notes in lab notebook