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

[DESCRIPTION]
<p>The use of genetically encoded ‘self-labeling tags’ with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved <em>Drosophila melanogaster</em> transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.</p>

[BEFORE_START]
<p><strong>Reagents and Supplies</strong></p>
<p><strong> </strong></p>
<ul>
<li>Corning dropping bottle (125 mL) with bulb and pipet. Fisher Scientific. # 02-986C</li>
<li>Cover glass 22x22 mm Square No. 1. Fisher Scientific.  # 12-542B (for mounting)</li>
<li>Cover glass 22x22 mm Square No. 2. Fisher Scientific.  # 12-540B (for spacers)</li>
<li>Cover glass staining jar.Electron Microscopy Sciences. # 72242-24 (4 pack)</li>
<li>Diamond Cleaver. com. # CLE</li>
<li>DPX Mountant for Microscopy. Electron Microscopy Sciences. # 13512, 500 mL</li>
<li>DPX Slide Jig.Available through Janelia Instrument Design &amp; Fabrication
<ul>
<li><a href="http://hhmi.flintbox.com/public/project/26605" target="_blank" rel="noopener noreferrer">http://hhmi.flintbox.com/public/project/26605</a></li>
</ul>
</li>
<li>Drosophila Mounting T-dish. Available through Janelia Instrument Design &amp; Fabrication.
<ul>
<li>designs available: 6x10 T-dish (for adult CNS), 8x10 (for adult brains) and larval T-dish.</li>
<li><a href="http://hhmi.flintbox.com/public/project/26606" target="_blank" rel="noopener noreferrer">http://hhmi.flintbox.com/public/project/26606</a></li>
</ul>
</li>
<li>ACS reagent,  &gt;99.5% (200 proof).  Sigma Aldrich. # 459844-1L</li>
<li>Kodak Photo-Flo 200 Solution. Electron Microscopy Sciences. # 74257</li>
<li>PBS – Phosphate Buffered Saline, 1X<strong>. </strong> # 21-040</li>
<li>PFA – Paraformaldehyde, 20% PFA. Electron Microscopy Sciences.  # 15713-S</li>
<li>Poly-L-Lysine.Sigma Aldrich. # P1524-25MG</li>
<li>Superfrost Plus Slides 25x75 mm. Fisher Scientific. #12-550-15</li>
<li>Tape – 0.5 in. wide. Fisher Scientific.  # 15-901-15A</li>
<li>Triton X-100. Sigma Aldrich. # X100</li>
<li>Xylenes. Fisher Scientific. # X5-500</li>
</ul>

[STEPS]
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TITLE: TS Spurrs - FFPE block - 1mm3 cube cut out - tissue (TM - 013)
AUTHORS: sandra.crameri

[DESCRIPTION]
Processing unstained parraffin block that has had a 1mm3 piece disected out of the face to Spurr's resin block for EM.

[GUIDELINES]
All step times are the minimum time required. Longer steps are acceptable.

[STEPS]
SECTION: HEADER
1. SAN:


SPEC No:


OPERATOR & STEPS:


OPERATOR & STEPS:


SECTION: Dewax FFPE sections
2.   or X3B 5 min at 60 °C 

SECTION: Dewax FFPE sections
3.   or X3B 5 min at Room temperature 

4.   or X3B 5 min at Room temperature 

SECTION: Rehydrate
5. 100 % volume   10 min 

6. 95 % volume    10 min 

7. 70 % volume   10 min 

SECTION: Conventional - with reduced times for 5um thick FFPE section
8. 2.5% % volume     in 0.1 Molarity (M) Sorenson's phosphate buffer pH 07.2 300 mosmol/kg20 min.

SECTION: Conventional - with reduced times for 5um thick FFPE section
9. Wash buffer 5 min 

SECTION: Conventional - with reduced times for 5um thick FFPE section
10. 
1 Mass / % volume   in buffer60 min 

SECTION: Conventional - with reduced times for 5um thick FFPE section
11. 70 % volume    10 min 

12. 95 % volume   10 min 

13. 100 % volume   10 min 

14. 100 % volume    10 min 

15. 100 % volume Spurrs:100 % volume    in 1:1 ratio30 min 

16. 100 % volume Spurrs60 min 

17. 100 % volume Spurrs60 min 

18. Oven polymerise 65 °C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div class="view-protocol-title vpt-block">
<p class="vpt-desc editable-content">For use in <a href="https://www.protocols.io/view/Generating-viral-metagenomes-from-the-coral-holobi-ejgbcjw" target="_blank">Generating viral metagenomes from the coral holobiont</a>.</p>
</div>

[STEPS]
?. 
?. 
?. 
?.
TITLE: Chromosomal DNA extraction from Gram-positive bacteria, V2
AUTHORS: Anders Kiledal, Julia A Maresca

[DESCRIPTION]
Extraction of high-molecular-weight DNA from Gram-positive bacterial species, with optional steps for removing surfactants. This DNA is suitable for sequencing and the protocol can be scaled up at least 5-fold. Modified from a protocol by Tina Wecke, LMU-Munich. 

[BEFORE_START]
Grow culture to high cell density and prepare all solutions. 

[GUIDELINES]
Recommend wearing gloves throughout and working in a biosafety cabinet if possible to prevent contamination. This protocol can be scaled up at least 5-fold. If the isopropanol precipitation step is used, subsequent steps do not have to be scaled up unless the culture volume is substantially larger.

[STEPS]
SECTION: Grow culture
1. Inoculate 10 mL rich medium from a fresh overnight culture, and incubate at appropriate temperature on shaker. At OD600 of ~0.8-1.0, harvest cells by centrifugation (10 min., 5000 rpm).
SECTION: Cell lysis
2. Resuspend cell pellet in 2 mL TEN (10 mM Tris-HCl, pH 8.0, 10 mM EDTA, 150 mM NaCl).
SECTION: Cell lysis
3. Add 100 μL lysozyme (20 mg/mL) and incubate for 20 min at 37ºC.
SECTION: Cell lysis
4. Add 20 μL RNAse (10 mg/mL) and incubate for 3 min at 65ºC.
SECTION: Remove surfactants (optional)
6. IF THE STRAIN PRODUCES A SURFACTANT THAT INTERFERES WITH THE PHASE SEPARATION, Add 0.1 volume 3 M sodium acetate and 1 volume cold isopropanol, mix, and incubate on ice for 20 min. Centrifuge for 10 min at 5000 rpm and decant the supernatant. Then resuspend in 400 μL TEN and 550 μL TEN* and transfer to a microcentrifuge tube.
SECTION: Phenol &amp; chloroform:isoamyl alcohol extractions
7. Add 900 μL phenol, mix by inversion. Centrifuge for 5 min. at 13000 rpm and transfer the upper phase to a clean microcentrifuge tube.
SECTION: Phenol &amp; chloroform:isoamyl alcohol extractions
8. Re-extract once with phenol (1 volume) and twice with chloroform: isoamyl alcohol (24:1 v/v, 1 volume)
SECTION: DNA precipitation
9. Collect DNA by coiling on the end of a glass Pasteur pipet.
SECTION: DNA precipitation
10. Air dry, then resuspend DNA in 100 μL sterile water overnight at 4ºC.
SECTION: Cell lysis
5. Add 40 µl SDS, a small scoop of proteinase K and 550 µl TEN*. Vortex, then incubate at 60°C for 2 hours.
TITLE: Fatty acid extraction and derivatisation
AUTHORS: iGEM Dusseldorf

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol enables direct, one-pot fatty acid extraction and derivatisation of plant and bacterial samples for preparation for GC-MS analysis.</div><div class = "text-block"><span>The protocol was established by the Weber lab for plant seedlings and seeds, but was successfully used for cyanobacteria and </span><span style = "font-style:italic;">E.coli</span><span> as well.</span></div></div>

[STEPS]
?. [Start]
samples with 4 oD-units ( e.g.: oD = 1, you will need 4 ml culture )you should make 4 or more replicates
?. [Start]
centrifuge in a clean glass tube @ 4500 x g
?. [Start]
discard supernatant
?. [Start]
Freeze @ -80 ˚C until further use
?. [Extraction solution]
200 µl C17 internal standard (1 mg/ml hexane) + 9,8 ml MeOH/3N HClcaution! fill in a beaker and take the needed amount out of this beaker, don't take out directly from original container to avoid contaminating stock solution.always work with gloves
?. [Extraction]
heat @ 90°Cafter 5 minutes re-tighten lids of the glass tubes!
?. [Extraction]
let cool down @ RT
?. [Extraction]
add 1 ml hexane
hexane is very volatile, that's why working quickly is necessary
?. [Extraction]
add 1 ml 1% NaCl
?. [Extraction]
vortex
?. [Extraction]
spin down @ 2000 rpm
?. [Extraction]
transfer hexane phase in to GC vial with screw cap
?. [Extraction]
dilute samples ( 10 µl sample + 90 µl hexane )wash hamilton between samples with hexaneall of these samples can be frozen @ -20 until further analysis
?. [Extraction]
add 1 ml extraction solution to samples and blankFor each new batch of samples, include a blank
TITLE: Longitudinal Analysis of C elegans (L4, YA D2 & YA D5)
AUTHORS: Priota Islam

[STEPS]
?. Bleach synchronize the worms following the protocol Bleach synchronisation of C. elegans
?. Refeed the arrested L1s 2 days prior to tracking (Refeed per strain on 3 plates)
?. Prepare the imaging plates by seeding with 50ul OP50 and leave to dry on bench top/hood
?. Image the strains at the following stages: L4, Day 2 and Day 5. On the day of imaging, pick 5 worms per imaging plate and place on the Phenix rig to acclimatize for 30mins followed by imaging for 15minutes
?. Discard the plates post imaging
?. Note:On D2 of the tracking, pick around 40 worms from each strain onto 2 new plates for D5 tracking, otherwise the plate will be too crowded, and it will be difficult to differentiate the age.
TITLE: Cylinder Test
AUTHORS: Sabina Marciano, Roberta Marongiu

[DESCRIPTION]
The Cylinder test is used to evaluate locomotor asymmetry in rodent models


[STEPS]
1. Place the mouse in the transparent cylinder for 5 min 

2. Place an angled mirror at the back of the cylinder to allow observation of behavior from every angle
3. Record mouse behavior for 5 min 

Main outcome measures: Left/Right contacts, Number of rears, L/R hind-paw contacts, Total time rearing, Time on the ground
TITLE: Chocolate Chip Cookies
AUTHORS: freddie.sherlock  

[DESCRIPTION]
This protocol outlines an optimised recipe for chocolate chip cookies, the recipe will make between 30 - 40 cookies.


[BEFORE_START]
Preheat fan oven to 180oC

[GUIDELINES]
Prepare ingredients before starting.

[STEPS]
SECTION: Dry Ingredient Preparation
1. In a bowl, mix the following amounts of dry ingredients to a large bowl and use a whisk to create a homogenous mix.
240 g
240 g 
10 g 
10 g 
265 g 
225 g 


SECTION: Wet Ingredient Preparation
2. Using a knife, slice 280 g into even chunks, transfer to a saucepan on low or medium heat and heat until melted.
SECTION: Wet Ingredient Preparation
4. Gently crack 2 large eggs into the large bowl of dry ingredients.

SECTION: Incorporation of Ingredients
5. Add the mixture containing 280 g and 10 g  to the large bowl and mix thoroughly.

SECTION: Wet Ingredient Preparation
3. Add 10 g  to the melted butter and mix thoroughly.

SECTION: Incorporation of Ingredients
6. Using the large knife and chopping board, cut 570 g into small chunks, approximately half the size of a cube section.

SECTION: Incorporation of Ingredients
7. Add 570 g to the large bowl and mix. This can be done with a wooden spoon, whisk or by hand as the mixture will be quite thick.

SECTION: Baking
11.  Add 8 ping-pong sized doughballs to a baking tray lined with parchment paper.


If baking from fresh, bake in the oven at 180oC for 8-10 minutes
If baking from frozen, bake at 180oC for 15-20 minutes.
SECTION: Incorporation of Ingredients
9. Shape the dough into small ping-pong ball spheres.
SECTION: Incorporation of Ingredients
8. Mixed dough can either be baked on the day or covered and frozen as 4 doughballs in a freezer bag at -20oC.
SECTION: Baking
12. Let the cookies sit for approximately 5 minutes, this is to both allow them to cool and to set.
SECTION: Incorporation of Ingredients
10. Use a small spoon to press in a dimple on the top of each ball.
SECTION: Baking
13. Enjoy cookies with some milk (or a milk substitute)!
TITLE: Melatonin ELISA
AUTHORS: daniel.dautan daniel, Per Svenningsson

[DESCRIPTION]
Measurement of mouse plasma melatonin using ELISA Kit (Enzo Life Sciences, ENZ-KIT150-0001, NY, US) according to manufacturer instructions.

[STEPS]
SECTION: Melatonin Extraction
1. Mix 200 µL of plasma with an equal volume of cold Ethyl Acetate. Vortex gently.
SECTION: Melatonin Extraction
2. Allow layers to separate on ice for 3 min. Vortex again and incubated on ice for 2 min. 
SECTION: Melatonin Extraction
3. Spin samples at 1000g for 10 min at 4 °C. Transfer the organic layer to a new tube. 
SECTION: Melatonin Extraction
4. Dry samples and resuspend in 220 µLof 1X stabilizer. 
SECTION: ELISA
5. Add 100 µL of standards' working solutions and samples  to provided 96-well plate in duplicates. 
SECTION: ELISA
6. Immediately after add 50 µL of melatonin tracer to each well (except blanks) followed by 50 µLof 1X melatonin antibody (except blanks). 

SECTION: ELISA
7. Cover plates with the provided plate sealer. Incubate for 60 min at 37 °C with 500 rpm shaking.
SECTION: ELISA
8. Decant the solution from each well. Add 400 µL of wash solution to each well. 
SECTION: ELISA
9. Decant the solution from each well and pat dry against clean absorbent paper. 
SECTION: ELISA
10. Repeat wash step 3 times.
SECTION: ELISA
11. Add200 µLof melatonin conjugate solution to each well (except blanks). Cover plate with the sealer. Incubate for 30 min at Room temperature.

SECTION: ELISA
12. Perform the wash step as described above. Add 200 µL of substrate reagent to each well. Incubate for 30 min at 37 °C protected from light. 

SECTION: ELISA
13. Add 50 µL of stop solution to each well. Measure the optical density using a micro-plate reader with absorbance set to 450 nm.
TITLE: Primer Preparation
AUTHORS: Dakota Betz

[DESCRIPTION]
Dilute primer stocks for use in your PCR reactions.

[BEFORE_START]
Use 75% Ethanol to clean the lab bench, tube racks, and pipettes before taking out any reagents and starting the protocol.

[STEPS]
SECTION: Clean lab area first!
1. Check that you have cleaned the lab bench, tube racks, and pipettes with 75% Ethanol before taking out any reagents and starting this protocol.
 

SECTION: Primer Preparation
3. Primer stocks are located in the -80 freezer (right side of shelf 2). Obtain only the primer stocks you need right now from the box, then place the box immediately back in the freezer to avoid thawing more primer stocks than necessary. Place the primer stocks you'll be using (forward and reverse) in your clean tube rack to thaw.
SECTION: Primer Preparation
2. Obtain a new water aliquot from the -20 frost-free freezer and place in your cleaned tube rack to thaw.
SECTION: Primer Preparation
4. While the stocks/water thaw, label one clean/new microcentrifuge tube for the forward primer and one for the reverse primer. Include the 1:10 concentration info, name of the primer, and today's date. 
SECTION: Primer Preparation
5. Use the following table to prepare the amount of stock you need. Aliquot water into your labeled microcentrifuge tubes first. Once the primers are thawed, be sure to mix them thoroughly (vortex) before pipetting the appropriate amount into your microcentrifuge tubes. Also be careful not to mix forward and reverse primers: use only one stock per microcentrifuge tube, labeled accordingly.
 
 

 
SECTION: Primer Preparation
6. Mix thoroughly (vortex) newly prepped primers. You can store these in your labeled PCR box in the -20 frost-free freezer. Be sure to return primer stocks to their appropriate box in the -80 as soon as you are finished.
TITLE: Imaging single AF647 molecules immobilised in PVA on a cover glass
AUTHORS: Ezra Bruggeman

[DESCRIPTION]
This is a protocol for the preparation of a microscopy sample of single Alexa Fluor 647 molecules immobilised in PVA polymer on a cover glass. This protocol was used to generate the data shown in Figure 2i and 1j of the following publication:

Bruggeman et al., POLCAM: Instant molecular orientation microscopy for the life sciences. bioRxiv 2023.02.07.527479 (Feb 2023), doi: https://doi.org/10.1101/2023.02.07.527479

[STEPS]
SECTION: Protocol
2. Argon plasma clean cover glass (VWR collection, 631-0124) for 30 min in a plasma cleaner (Expanded Plasma Cleaner, PDC-002, Harrick Plasma).
SECTION: Protocol
1. Prepare a 1% poly(vinyl) alcohol (PVA) solution:
SECTION: Protocol
3. Dilute Alexa Fluor 647 (AF647) to 500 picomolar (pM) in the filtered 1% PVA solution.
SECTION: Protocol
1.2. Dissolve 1 g of solid PVA into 100 mL of filtered milli-Q water in a glass beaker. 

SECTION: Protocol
1.1. Filter 100 mLMilli-Q water using a syringe filter (0.02 μm syringe filter, Whatman, 6809-1102).
SECTION: Protocol
1.4. Let the solution cool. Once cooled, filter it using a syringe filter (0.02 μm syringe filter, Whatman, 6809-1102). You can store the filtered 1% PVA solution at 4 °C for later use.
 

SECTION: Protocol
4. Place a clean cover glass into a spincoater (WS-650Mz-23NPPB, Laurell) and spin-cast 10 µL of the AF647 solution onto the cover glass for 45 s at 3000 rpm.
 

SECTION: Protocol
5. Optional: Seal the sample by sticking it to a standard glass microscope slide (631-1550, VWR) using a double-sided frame-seal slide chamber sticker (9x9 mm, SLF0201, Bio-rad). Make sure the coated side of the cover glass is facing the microscope slide.
SECTION: Protocol
1.3. Place the beaker on a stirring hot plate and heat the solution to 90 °C under continuous stirring for 30 min or until the PVA is completely dissolved.
TITLE: JAX-Sen: Mouse kidney dissociation for single-cell RNA sequencing
AUTHORS: Ramalakshmi Ramasamy, Juliana Alcoforado Diniz, Jessica Garofalo, Patrick Fleming, Paul Robson

[DESCRIPTION]
These samples are part of the JAX-Sen project in the SENNET Consortium. We aim to study and characterize senescence in the C57Bl/6 mouse kidney. We compare young (6 months old) and old (24 months old) mouse kidneys using scRNAsequencing. This protocol describes the single-cell dissociation of the kidneys before library preparation and sequencing.

[STEPS]
SECTION: Reagents and Materials
1. 5 ml Protein lobind tubes
1.5 or 2 ml Protein lobind tubes
Micro scissors/ scalpel
Ice-cold PBS
Wide-bore pipette tips
GentleMACS dissociator
GentleMACS C tubes
DMEM + 10% FBS
ACK lysis buffer – 2 ml/sample
SS_buffer– 5 ml/sample
40um cell strainer
70uM strainer

SS_Buffer:

 

 
SECTION: Procedure
2. Collect shipment from the warehouse.
SECTION: Procedure
2.1. Fill up ice buckets and prep them with reagents:
Bucket 1(small): Enzyme mix, PBS, SS_buffer
Bucket 2 (large): For mincing tissues and sample tubes
SECTION: Procedure
2.2. Confirm if the sample is cold. Take pictures. Transfer sample to 5 ml tubes.
SECTION: Procedure
2.3. Prepare enzyme mix by adding 2.35 mL of serum-free RPMI 1640 or DMEM, 100 µL of Enzyme D, 50 µL of Enzyme R, and 12.5 µL of Enzyme A of the Multi Tissue Dissociation Kit 1 into a gentleMACS C Tube for up to 0.5 g of tissue. 
SECTION: Procedure
2.4. Mince mouse kidneys (2-4 mm3) with sterile micro scissors.
SECTION: Procedure
2.5. After cutting, transfer the sample into labeled 5 ml tubes. 
SECTION: Procedure
2.6. Tightly close C Tube and attach it upside down onto the sleeve of the gentleMACS Dissociator.
SECTION: Procedure
2.7. Run the gentleMACS Program Multi_B. If using the heating function of the gentleMACS Octo Dissociator with Heaters run program 37C_Multi_E. 
SECTION: Procedure
2.8. After termination of the program, detach C Tube from the gentleMACS Dissociator.
SECTION: Procedure
2.9. Resuspend sample and apply the cell suspension to a MACS‱ SmartStrainer (70 µm) placed on a 5 mL tube.
SECTION: Procedure
2.10. Wash MACS SmartStrainer (70 µm) with 4 mL of ice-cold PBS.
SECTION: Procedure
2.11. Centrifuge at 300 x g, 4°C, 7 min to get rid of the supernatant. Aspirate supernatant.
SECTION: Procedure
2.12. Resuspend the cell pellet in 2 mL  ACK Lysing buffer, and incubate on ice for 3 min to exclude RBCs.
SECTION: Procedure
2.13. Add 5 mL SS_buffer  to stop  ACK Lysing buffer reaction, then collect cells by centrifugation 500 x g, 4°C, 5 min.
SECTION: Procedure
2.14. Resuspend the pellet in 1ml of ice cold PBS.
SECTION: Procedure
2.15. Filter the cell suspension through  Flowmi 40um cell strainers tube to remove debris and non-dissociated tissue fragments. 
SECTION: Procedure
2.16. Collect cells by centrifugation 500 x g, 4°C, 5 min and proceed to Flex protocol.
TITLE: Making OP50 solution from Frozen Stock
AUTHORS: Priota Islam

[STEPS]
?. Day: 1 Get LB Agar from the media kitchen and autoclave it for 2hrs Post autoclave, pour the agar on large petri dishes (60mm) and leave to dry overnight to be transferred to the cold room the next day
?. Day: 2 Get the frozen tube out of the freezer (Freezer 12, SD Box) Take a sterile pipette tip and get some frozen sample  Streak the pipette on the LB agar plate Take another new pipette and streak across the old streak and then at a separate spot streak again Repeat this step for one more time Keep the plate at 37°C incubator overnight for the bacteria to grow Transfer the plate to the fridge the next day (This plate can be used for about 1 month to inoculate bacterial culture)
?. Day:3  Purchase LB Broth from the Media kitchen (Don’t use less than 200ml) Get flat bottomed conical flasks from the glassware kitchen (For 200ml LB Broth take 400/500ml flask to allow enough headspace for the bacteria) From the culture plate, select and circle a single colony  Using aseptic technique, carefully scoop the single colony and mix it with the LB Broth that is already poured into the conical flask Label the flask and put it on the 37°C shaker for overnight incubation
?. Day:4  Following overnight incubation, take the flask out of the incubator and measure the OD using a spectrophotometer Record the average of the OD600, use LB Broth as Blank Aliquot the solution into labelled 15ml falcon tubes and keep them in the 4°C Fridge
TITLE: Preparation of feeder-free iPSCs culture
AUTHORS: Celeste Karch, Rita Martinez, Jacob Marsh

[STEPS]
?. Coat appropriate sized tissue culture dish with Matrigel. Swirl plate to distribute evenly across surface of dish.
?. Allow Matrigel to set for at room temperature or in a humidified chamber.
This will provide enough cells for a full 96 well plate of neural aggregates.
We typically thaw 1 vial of iPSC into 1 well of a 6 well (1mL Matrigel per well). Volumes below are based on 6 well plates.
?. Prepare mTeSR1 feeder-free medium by aseptically adding  of thawed 5x supplement to Basal Medium.
100 ml
400 ml
?. Add penicillin/streptomycin if needed. Mix thoroughly and warm to room temperature.
5 ml
?. Thaw iPSCs (1-2 x 106 cells/mL) in a  water bath.
?. Add  of thawed iPSC to of DMEM/F12 in a 15mL conical tube (Do not mix). Centrifuge iPSC at 750 rpm for .
1 ml
9 ml
?. Carefully aspirate medium from iPSC pellet. Add of mTeSR1 supplemented with Rock inhibitor (10 μM final) - avoid breaking up clumps of suspended cells.
2 ml
?. Aspirate Matrigel from tissue culture dish. Add  of suspended iPSCs. Place plate in , 5% CO2 and 95% humidified chamber, making a “T” motion to ensure even distribution of cells.
2 ml
?. Change medium with  of fresh mTesR1 medium daily.
2 ml
As iPSC become more confluent, increase mTesR to 3mL per well.