eamag/protocols-with-steps huggingface dataset:
TITLE: Yale Murine TMC - H&amp;E staining  following Phenocycler-Fusion Imaging
AUTHORS: Jungmin Nam, Yale Pathology Tissue Services, Rong Fan, Archibald Enninful

[DESCRIPTION]
Flow cell removal after Phenocycler-Fusion imaging and following H&E staining steps from Yale Pathology Tissue Services (YPTS).

[STEPS]
SECTION: Hematoxylin and eosin staining
6. Stain with hematoxylin for 5 min. 
SECTION: Hematoxylin and eosin staining
10. Bluing for 1 min. 
SECTION: Hematoxylin and eosin staining
9. Rinse with water. 
SECTION: Hematoxylin and eosin staining
8. Clarifier for 30 seconds. 
SECTION: Hematoxylin and eosin staining
7. Rinse with water. 
SECTION: Hematoxylin and eosin staining
11. Rinse with water. 
SECTION: Hematoxylin and eosin staining
12. Stain with eosin 5 min. 
SECTION: Hematoxylin and eosin staining
13. Immerse slides in graded ethanol to xylene.
SECTION: Hematoxylin and eosin staining
14. Add coverslip with resin mounting medium.
SECTION: Removing flow cell following CODEX imaging
1. Remove slides from storage buffer.
SECTION: Removing flow cell following CODEX imaging
2. Immerse slides to xylene for 10 mins.
SECTION: Removing flow cell following CODEX imaging
3. Separate flow cell from the slide using a razorblades.
SECTION: Removing flow cell following CODEX imaging
4. Rinse slide with 1x PBS.
SECTION: Removing flow cell following CODEX imaging
5. Store in storage buffer until H&E staining.
TITLE: Increased sensitivity of Euplotes crassus to selective agents using 0.3 M glucose-based culture conditions.
AUTHORS: Lawrence A. Klobutcher, Larry Klobutcher

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Numerous unpublished studies indicate that </span><span style = "font-style:italic;">Euplotes crassus</span><span> is resistant to extremely high concentrations of commonly used selective agents when grow in artificial seawater. We have found that when </span><span style = "font-style:italic;">E. crassus</span><span> is grown in 1 part artificial seawater + 9 parts 0.3 M glucose, they are much more sensitive to a number of selective agents. For example, 1080 ug/ml paromomycin in artificial seawater only inhibited growth, as little as 120 ug/ml was effective in killing cells when grown with 0.3 M glucose. Similarly, G418 at 400 ug/ml had little effect in seawater, but as little as 100 ug/ml blocked growth in 0.3 M glucose. This may not prove true for all selective agents, as we saw no effect with paclitaxel up to 100 uM under either growth condition.</span></div></div>

[STEPS]
?. Distribute a dense culture of Dunaliella salina to two 50 ml screw-cap centrifuge tubes, and pellet the algae by centrifugation at 1,000 RPM (~200 x g) for 2 min. in a clinical centrifuge.
?. Remove all but the last 2.5 ml of supernatant from each tube, resuspend the algae pellets, and combine.
?. To the above tube, add 2.5 ml of a log phase culture of Euplotes crassus (grown with Dunaliella in artificial seawater as the food source), and 45 ml of 0.3 M glucose (freshly prepared using deionized water).
?. Add selective agent to desired final concentration. 200 ug/ul G418, 200 ug/ml paromomycin, or 40 ug/ml puromycin.
?. Cells can be distributed microtiter plates. For example, 2 ml per well for a 24-well microtiter plate.
?. Incubate at room temperature in a humidified chamber (e.g., plastic box with wet paper towels or beaker of water). Cell death occurs within 2-3 days.
?.
TITLE: Preparing 10x TBE Electrophoresis buffer
AUTHORS: Nadine    Mowoh, Jenny  Molloy

[DESCRIPTION]
TBE and TAE buffers are often used in procedures involving nucleic acids, the most common being electrophoresis. Tris-acid solutions are effective buffers for slightly basic conditions, which keep DNA deprotonated and soluble in water. 

EDTA is a chelator of divalent cations, particularly of magnesium (Mg2+). As these ions are necessary cofactors for many enzymes, including contaminant nucleases, the role of the EDTA is to protect the nucleic acids against enzymatic degradation. But since Mg2+ is also a cofactor for many useful DNA-modifying enzymes such as restriction enzymes and DNA polymerases, its concentration in TBE or TAE buffers is generally kept low (typically at around 1 mM).

TBE buffer is used  to prepare agarose gels and as running buffer in agarose gel electrophoresis to separate and identify and analyse nucleic acids.

 SCOPE:

This protocol covers the steps involved in making 1L of 10x TBE buffer from Tris, Borate and EDTA powders

 DEFINITIONS:

TBE: Tris Borate EDTA


[GUIDELINES]
This procedure can be performed by laboratory staff that have been trained and have theoretical and practical skills in good laboratory practices. It can also be performed by molecular biology students or students of related fields under the supervision of a laboratory staff.

[STEPS]
SECTION: Preparing 10x TBE buffer solution (1 liter)
1. Use an electronic balance to weigh out 108 g Tris base (CAS# 77-86-1, free base) into a 1000 mL ml or higher volume beaker ( depending on what volume of beaker is available and should be big enough to contain the powders to be dissolved).
SECTION: Preparing 10x TBE buffer solution (1 liter)
2. Weigh 55 g boric acid (CAS# 10043-35-3) into the same beaker
SECTION: Preparing 10x TBE buffer solution (1 liter)
5. Adjust volume to 1 L by adding 900 mL of distilled or deionised water.
SECTION: Preparing 10x TBE buffer solution (1 liter)
4. Measure 100 mL  distilled or deionised water using a measuring cylinder, pour into the 1000 mL beaker and stir for 1 min either manually or using a magnetic stirrer and flea.
 


SECTION: Preparing 10x TBE buffer solution (1 liter)
3. Weigh9.3 g EDTA, disodium salt dihydrate (Cas# 6381-92-6, C10H14N2Na2O8· 2H2O, MW: 372.24) into a weighing boat and pour into the beaker.
 

SECTION: Preparing 10x TBE buffer solution (1 liter)
6. Place the beaker on top of a magnetic stirrer and put a magnetic flea in the solution. Allow to stir until you have a clear solution, this may take up to 30 min 
SECTION: Preparing 10x TBE buffer solution (1 liter)
8. Autoclave (if sterile solution is needed) and pour solution into clean, autoclaved and labelled glass bottles.
 

SECTION: Make 1 x TBE working solution
9. This buffer can be diluted to 1x each time before use by diluting 100 mL  of 10x TBE with 900 mL of distilled water to have 1 L  of 1x TBE buffer.
SECTION: Preparing 10x TBE buffer solution (1 liter)
7. Check the pH using a pH meter or Universal indicator strips and record the result for quality control. A properly prepared solution should be pH 8.3 and should not require adjustment.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol was developed for isolating high quality genomic DNA from Heterosigma akashiwo for the purpose of Next generation sequencing technologies. We successfully repeated this protocol to obtain genomic DNA.  </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Purification of NIX-GFP
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol details the purification of NIX-GFP.

[STEPS]
SECTION: Purification - NIX-GFP
1. To purify GFP-tagged 

NIX-GFP (available from Addgene) or NIX(W36A/L39A)-GFP (ΔLIR)(available from Addgene), 

fuse the cytosol-exposed domain of NIX (1-182aa) to a C-terminal GFP-tag through cloning into a pET-DUET1 vector (available from Addgene). 
SECTION: Purification - NIX-GFP
2. Introduce the point mutants in vitro mutagenesis to generate 

NIX E72A/L75A/D77A/E81A (4A; ΔWIPI2) (available from Addgene), and 
NIX W36A/L39A (ΔLIR) (available from Addgene).
SECTION: Purification - NIX-GFP
3. After the transformation of the pET-DUET1 vector encoding NIX-GFP wild-type or mutants in E. coli Rosetta pLysS cells (Novagen Cat# 70956-4), grow the cells in 2x Tryptone Yeast extract (TY) medium at 37 °C until an OD600 of 0.4 and then continue at 18 °C. 
SECTION: Purification - NIX-GFP
4. Once the cells reaches an OD600 of 0.8, induce the protein expression with  100 micromolar (µM) isopropyl β-D-1-thiogalactopyranoside (IPTG) for 960 min at 18 °C. 
SECTION: Purification - NIX-GFP
5. Collect the cells centrifugation and resuspend in lysis buffer. 

 Lysis buffer:
 

SECTION: Purification - NIX-GFP
6. Sonicate the cell lysates twice for 30 s and clears by centrifugation at 18.000 rpm, 45 min, 4 °Cin a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific). 
SECTION: Purification - NIX-GFP
7. Collect the supernatant and incubate with pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 120 min at 4 °C with gentle shaking to bind NIX-GFP. 
SECTION: Purification - NIX-GFP
6.1. Sonicate the cell lysates for 30 s (1/2). 
SECTION: Purification - NIX-GFP
6.2. Sonicate the cell lysates for 30 s (2/2). 

SECTION: Purification - NIX-GFP
8. Centrifuge the samples to pellet the beads and remove the unbound lysate. 
SECTION: Purification - NIX-GFP
9. Wash the beads twice with wash buffer, once with high salt wash buffer, and two more times with wash buffer. 

 Wash buffer: 

 

High salt wash buffer:
 
 
 
SECTION: Purification - NIX-GFP
10. Cleave off the GST-tag   by eluting the GFP-tagged cargo receptor from the GSH beads by the addition of TEV protease in wash buffer at 4 °C.

 Wash buffer: 
 
 
 
SECTION: Purification - NIX-GFP
11. To collect the supernatant, collect the beads by centrifugation. 
SECTION: Purification - NIX-GFP
12. Wash the beads twice with 4 mL of wash buffer, and collect the supernatant. 
SECTION: Purification - NIX-GFP
13. Pool the supernatant fractions, filter through a 0.45 µm syringe filter, concentrate with 10 kDa cut-off Amicon filter (Merck Millipore), and load onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva). Elute the proteins with SEC buffer. Analyze the fractions by SDS-PAGE and Coomassie staining. Pools fractions containing purified NIX-GFP. 

 SEC buffer:

 
 
SECTION: Purification - NIX-GFP
14. After concentrating the purified protein, aliquote the protein and snap-frozen in liquid nitrogen.
TITLE: Isolate prokaryotes from sponge tissue (SAP)
AUTHORS: Martin Thomas Jahn

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Protocoll to create a fixed suspension of sponge associated prokaryotes</span><span> (SAP) from sponge tissue.This can serve as the basis for Fluorescence </span><span style = "font-style:italic;">in situ </span><span>hybridisation and/or cell sorting.</span></div><div class = "text-block">This protocol was tested for different sponge species but might also be adapted to other organisms (let others know)</div><div class = "text-block">The protocol was modified from :</div><div class = "text-block">Fieseler L, Horn M, Wagner M, Hentschel U. (2006) Discovery of the novel candidate phylum "Potibactetia" in marine sponges (vol 70, pg 3724, 2004). Applied and Environmental Microbiology;72(8):5677-.</div><div class = "text-block"><span>PMID:15184179 DOI:</span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">10.1128/AEM.70.6.3724-3732.2004</span></a></div></div>

[STEPS]
?. [Dissect and homogenise tissue]
Mince sponge tissue in ice cold CMASW using a razor blade within a petry dish on ice
Tissue can be either fresh or thawed from -20°C samplesProcess about the tissue volume that would fit in a 1.5 ml eppendorf tube
?. [Dissect and homogenise tissue]
Squeeze remaining pieces using 15 ml falcon tube lid
[on ice]
?. [Dissect and homogenise tissue]
Vortex strongly at
21 °C
?. [Dissect and homogenise tissue]
Transfer to fresh 50ml Falcon tube and add up to
[CMASW]
?. [Dissect and homogenise tissue]
Incubate  on ice
?. [Purify]
Filter through 40µm cell strainer into a fresh 50 ml Falcon tube
?. [Purify]
Transfer to fresh 50 ml falcon tube and add up to
[CMASW]
?. [Purify]
Spin down at 600g to pellet cells for at
4 °C
?. [Purify]
Transfer supernatant to fresh 50ml falcon tube and add up to
[CMASW]
The supernatant should contain the bacterial fraction
?. [Purify]
Spin down at 1600g to pellet cells for at  and resuspend in
4 °C
[CMASW]
?. [Purify]
Repeat step 10 until solution becomes clear
In case of sponge tissue this gets rid of secondary metbolites that inhibit downstream applications.
?. [Fix cells]
If solution is clear resuspend pellet into 1ml ice cold  and transfer to fresh 2ml tube
[PFA in CMASW]
{"blocks":[{"key":"7krhm","text":"Fixative for FISH and other applications","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}
?. [Fix cells]
Fix overnight
[in fridge]
Fixation time and fixative concentration might be optimised for different cell typesfind basic guidelines here: Silva protocols
?. [Fix cells]
Pellet cells by centrifugation () discharge supernatant
[at 4000 x g]
?. [Fix cells]
Thoroughly resuspend fixed cells in 500 µl PBS
?. [Fix cells]
Repeat step 5 and 6
?. [Fix cells]
Add 500 µl absolute ethanol and resuspend cells thoroughly
?. [Fix cells]
At this stage the cell suspension can be stored at   for several months
-20 °C
TITLE: Sequence Analysis of a Plasmid
AUTHORS: Addgene The Nonprofit Plasmid Repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is for sequence analysis of a plasmid. To see the full abstract and additional resources, please visit </span><a href="https://www.addgene.org/protocols/sequence-analysis" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.addgene.org/protocols/sequence-analysis/</span></a><span style = ":;">.</span></div><div class = "text-block"><span style = "font-weight:bold;">Sample data</span></div><div class = "text-block">A good sequencing reaction will produce between 300-900 base pairs of useable sequence. You should receive your sequencing results as a trace file (.ab1) which graphically depicts the sequence as a series of colored peaks corresponding to one of the four nucleotide bases. This is an example of a trace file from a high-quality portion of a sequencing reaction:</div><div style = "text-align :; float : ;"><img style = "" src = "https://media.addgene.org/data/easy-thumbnails/filer_public/cms/filer_public/4d/f8/4df8a318-6c00-4465-a2ba-83e5541460f8/clean_sequence.jpg__260x128_q85_subsampling-2_upscale.png" /></div><div class = "text-block">Sequence near the beginning or end of a sequencing reaction is often unreliable. Although your sequencing results may indicate bases at specific locations, by looking at the trace file, you will see that these base calls are unreliable. This is an example of a trace file from a portion of a sequencing reaction with high background:</div><div style = "text-align :; float : ;"><img style = "" src = "https://media.addgene.org/data/easy-thumbnails/filer_public/cms/filer_public/b3/24/b324b8a7-bc8b-408c-a4fd-e1fc19cae8a5/high_background_sequence.jpg__260x123_q85_subsampling-2_upscale.png" /></div></div>

[STEPS]
TITLE: Prepare NGM plates with fungzizone
AUTHORS: Research Cancer UK / Wellcome Gurdon Institute media kitchen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Prepare NGM plates with fungizon</div></div>

[STEPS]
?. 
?. ABCD1Ingredients  Quantity2NGM Media  1L3Cholesterol 5mg/ml1ml41M CaCl21ml51M MgSO41ml61M KH2PO425ml7Petri dish30mmas required8 50mmas required9 90mmas required10Fungizone400 microlitres 1 microtubbe
ABCD1Ingredients  Quantity2NGM Media  1L3Cholesterol 5mg/ml1ml41M CaCl21ml51M MgSO41ml61M KH2PO425ml7Petri dish30mmas required8 50mmas required9 90mmas required10Fungizone400 microlitres 1 microtubbe
?. A1First thing in the morning, melt 1L NGM in
  autoclave2Once melted, place in water bath set at 58oC, leave until cooled
  (-at least 2hours) normally left until the afternoon.3Set out the plates on bench before starting
  pouring process. 
    Put melted NGM media on stirrer
    4Using 1ml gilson add&nbsp; the Cholesterol,replace the lid and return
  to the bench to give a good stir to dispense all the cholesterol.5Add the KH2PO4 using sterile 50ml syringe and 0.22µm filter.6Stir for a few seconds until well mixed. Add
  Fungizone ensuring all is added to the NGM.7Dispense media using a peristaltic pump. Add
  4.20ml in 30mm plates, 9.00ml in 50mm plates, 4.50ml in 6 well plates and
  2.5ml in 12 well plates. When set stack upside down in plastic trays.
A1First thing in the morning, melt 1L NGM in
  autoclave2Once melted, place in water bath set at 58oC, leave until cooled
  (-at least 2hours) normally left until the afternoon.3Set out the plates on bench before starting
  pouring process. 
    Put melted NGM media on stirrer
    4Using 1ml gilson add&nbsp; the Cholesterol,replace the lid and return
  to the bench to give a good stir to dispense all the cholesterol.5Add the KH2PO4 using sterile 50ml syringe and 0.22µm filter.6Stir for a few seconds until well mixed. Add
  Fungizone ensuring all is added to the NGM.7Dispense media using a peristaltic pump. Add
  4.20ml in 30mm plates, 9.00ml in 50mm plates, 4.50ml in 6 well plates and
  2.5ml in 12 well plates. When set stack upside down in plastic trays.
TITLE: TaqMan SNP genotyping protocol
AUTHORS: Heather Robeson, Jing Jin, Mohammed S. Orloff

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The protocol is based on TaqMan SNP genotyping protocol</div></div>

[STEPS]
?. [PCR reactions]
384-well Fast (5‐μL reaction ) Prepare the reaction mix:Combine the following components for the number of reactions required, plus 10% overage. 2X TaqMan Master Mix:   20X Assay Working Stock: Total volume per well:
2.5 µl
0.25 µl
2.75 µl
?. [PCR reactions]
Vortex to mix
?. [PCR reactions]
Centrifuge to bring the reaction mix to the bottom of the tube and remove the air bubbles
?. [PCR reactions]
Prepare the reaction plate:Dilute each DNA sample, positive and negative controls in Nuclease-free water. The final concentration should be at least 0.2 ng/μL
?. [PCR reactions]
Add reaction mix to each well of the reaction plate
?. [PCR reactions]
Seal the plate with adhesive film, then centrifuge to bring the reaction mix to the bottom of the well and eliminate air bubbles.
?. [PCR reactions]
Remove film from the plate, then add the appropriate volume of sample or control to the wells
?. [PCR reactions]
Seal the plate with adhesive film, then centrifuge to bring the reaction mix to the bottom of the well and eliminate air bubbles.
?. [Perform PCR]
Place the plate in a Real-Time PCR instrument ABI 7900 HT. Use the thermal cycling conditions specified.Polymerase activation: Temp.  , Time 10mins , Cycles HOLDDenaturation:Temp.  , Time 15 seconds, Cycles 40Annealing extension:Temp.  , Time 1 min, Cycles 40
95 °C
95 °C
60 °C
?. [Post-PCR plate read and analyses]
TITLE: Fragment Analyzer Operation for PCR Products
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol describes fragment analyzer operation for polymerase chain reaction (PCR) products. The Fragment Analyzer by Advanced Analytical is used as a quality control step for genotyping PCR products.  Peak size, shape, and sample concentration are recorded and used to evaluate samples on a pass/fail level.

Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

[STEPS]
TITLE: NCEM Drop conventional (TM-014)
AUTHORS: sandra.crameri

[DESCRIPTION]
Standard routine conventional negative contrast EM.

[STEPS]
SECTION: HEADER
1. SAN:




SPEC No:




OPERATOR:



SECTION: Conventional
2. Adsorb 10 µL sample to grid 10 min , visually inspect to ensure sample does not dry out in this time.

SECTION: Conventional
3. Drain excess sample from grid using filter paper, leave wet.
SECTION: Conventional
4. Stain  1 min 

5. Drain & dry using filter paper
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol gives a method for treatment and preservation of seawater samples that will be subject to fluorescence in situ hybridization (FISH).&nbsp;Samples are used with fluorescent probes to determine what populations of bacteria or other microbes are present. The filters may also be used to simply count the number of microbes in a sample.&nbsp;Requires varying amounts of 10&micro;m or 200&micro;m filtrate (up to 11.5L for open ocean depth profile) as shown in Table I.

[BEFORE_START]
Have fresh, unopened bottle of 37% formaldehyde on hand (see Table 1 to determine amount of reagent needed).

[GUIDELINES]
Requires varying amounts of 10&micro;m or 200&micro;m filtrate (up to 11.5L for open ocean depth profile) as shown in Table I.<br /><br />Table I. Samples from open ocean depth profile for FISH. Goal is for cell concentrations of about 10<sup>6</sup>/ml.<br /><br />
<table>
<tbody>
<tr>
<td><strong>Depth</strong></td>
<td><strong>Volume Seawater</strong></td>
<td><strong>Volume 37% Formaldehyde</strong></td>
<td><strong># of Reps</strong></td>
</tr>
<tr>
<td>10m</td>
<td>150ml</td>
<td>8ml</td>
<td>2</td>
</tr>
<tr>
<td>500m</td>
<td>1000ml</td>
<td>54ml</td>
<td>2</td>
</tr>
<tr>
<td>1000m</td>
<td>2000ml</td>
<td>108ml</td>
<td>2</td>
</tr>
<tr>
<td>2000m</td>
<td>2600ml</td>
<td>140ml</td>
<td>2</td>
</tr>
<tr>
<td>Total with reps</td>
<td>11.5 L</td>
<td>620ml</td>
<td>8 filters</td>
</tr>
</tbody>
</table>
<br />Formaldehyde is not a stable reagent.&nbsp; For this reason, use a fresh, unopened bottle of 37% formaldehyde for fixing samples.&nbsp; Use the table above to determine how much reagent you will need.

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: How to make a cup of tea
AUTHORS: ines.boehm 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is part of the </span><a href="https://carpentries-incubator.github.io/fair-bio-practice/06-record-keeping/index.html" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Record Keeping</span></a><span> lesson within the </span><a href="https://carpentries-incubator.github.io/fair-bio-practice/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">FAIR in (biological) practice</span></a><span> DataCarpentry course. Here we explore the perfect protocol to brew tea.</span></div></div>

[STEPS]
?. [Preparation of water and tea]
Fill the kettle with ~250mL of water, or until your min fill line and boil the water.
?. [Preparation of water and tea]
In the meantime prepare your tea bag, unpack it and place it in the cup/mug so that the tag is positioned outside of the mug.
?. [Preparation of water and tea]
Once the water is boiled, instantly pour the hot water into the mug. Ensure you fix the tag on the outside of the mug with your finger, to avoid falling in of the string + tag of the tea bag. Otherwise removal of the tea bag becomes more difficult.
?. [Brewing]
Brew the tea for 3 min
?. [Brewing]
Use the string/tag to remove the tea bag. Discard the tea bag in your compost for most appropriate recycling.
?. [Flavouring]
Add 5 mL = 1 teaspoon full fat milk to your tea
?. [Flavouring]
Enjoy!