eamag/protocols-with-steps huggingface dataset:
TITLE: Microplastic SEM Sample Prep
AUTHORS: Melissa B. Duhaime, Rachel Cable

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. [Sample Dehydration]
Fill a petri dish with enough PBS to submerge your microplastic sample. Rinse the plastic in the PBS bath to remove loosely associated debris.
?. [Drying]
Air-dry the dehydrated samples overnight in desiccator underneath a fume hood.
[overnight]
?. [Affix]
Affix plastic to double-side carbon tape on aluminum stud. Draw a strap of carbon paint from stud base to top of plastic. Dry overnight in desiccator.
[overnight]
?. [Microscopy ]
The next day, sputter-coat plastic sample with heavy metal.
?. [Sample Dehydration]
Next, create similar baths of ethanol or HMDS, as indicated below. Submerge each piece of plastic in the bath, let incubate in solution for  Washes:25% ETOH50% ETOH 70% ETOH95% ETOH100% ETOH100% ETOH2 ETOH: 1 HMDS1 ETOH: 1 HMDS1 HMDS
?. [Microscopy ]
Image sample under scanning electron microscope as soon as possible following sputter-coating.
Samples keep for a maximum of 2 weeks after prep.
TITLE: Cell Culture Reagents and Sources – 2023-08-30
AUTHORS: Vipasha Dwivedi, David J Riese II

[DESCRIPTION]
The COVID-19 pandemic and its aftermath have created additional challenges in maintaining the supply chain to support biomedical research laboratory operations.  A related challenge is recording and sharing cell culture conditions and reagents among laboratory members.  Through this protocols.io publication, we attempt to address both of these challenges.  

[STEPS]
1. The COVID-19 pandemic and its aftermath have created additional challenges in maintaining the supply chain to support biomedical research laboratory operations.  A related challenge is recording and sharing cell culture conditions and reagents among laboratory members.  Through this protocols.io publication, we attempt to address both of these challenges.  The spreadsheet associated with this publication   describes the media, sera, and other supplements used for propagating the cell lines in our laboratory.  The spreadsheet includes the concentration of G418, hygromycin, blasticidin, and puromycin used for selecting stable recombinant derivatives of these cell lines.  Finally, the spreadsheet contains the current vendor and catalog number for these cell culture reagents.

2. This manuscript will be revised and republished as we change our standard operating procedures.  To assist readers in identifying the current version of this manuscript, we will include the publication date in the title.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>Materials:</strong><br /><br />
<ol>
<li>Glass petri plates of NC64A chlorella lawns with virus plaques (use plates that have been incubating for 3-4 days)</li>
<li>Nitrocellulose circles</li>
<li>Tris buffered saline + Tween 20 (TBST, 1X): 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20</li>
<li>Blocking solution:  1% BSA in 1X TBST</li>
<li>Alkaline phosphatase buffer (AP, 1X):  100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl<sub>2</sub></li>
<li>Reaction stop/storage buffer (1X):  20 mM Tris-HCl, pH 8.0, 5 mM EDTA</li>
<li>Primary (1°) antiserum:  Chlorella virus antiserum, produced in rabbits</li>
<li>Secondary (2°) antiserum:  Anti-rabbit IgG alkaline phosphatase conjugate</li>
<li>NBT substrate:  Nitro blue tetrazolium</li>
<li>BCIP substrate:  5-bromo-4-chloro-3-indolyl phosphate</li>
<li>Glass petri dishes</li>
<li>Rotary shaker</li>
<li>Reaction mixture:  0.33 mg/mL NBT + 0.17 mg/mL BCIP in 1X AP buffer.  Dissolve the BCIP in 500 µL of dimethylformamide, then add dropwise to the NBT dissolved in the AP buffer</li>
</ol>

[STEPS]
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?.
TITLE: No Title
AUTHORS: 

[STEPS]
SECTION: Prepare individual amino acid stock solutions
?. 
SECTION: Prepare amino acid mix solution (∼17 nM each)
?. 
SECTION: Prepare maltodextrin-based energy solution
?. 
SECTION: Prepare maltodextrin-based energy solution
?. 
SECTION: Prepare amino acid mix solution (∼14 mM each)
?. 
SECTION: Prepare maltodextrin-based energy solution
?. 
SECTION: Prepare hexametaphophate solution
?.
TITLE: KAPP-Sen TMC: Pancreas Donor Acceptance Criteria
AUTHORS: Cristina Aguayo-Mazzucato, Kanako Iwasaki

[DESCRIPTION]
Pancreas Donor Acceptance Criteria

[STEPS]
SECTION: Inclusion Criteria
1. Age groups:
5 donors of age under 35
10 donors of age at least 35 and under 60
10 donors of age at least 60
SECTION: Exclusion Criteria
2. Diabetes mellitus
BMI > 30
Pancreatitis
Pancreatic cancer or metastasis
TITLE: KAPP-Sen TMC: Dissociation of Pancreatic Islets (recovered) Protocol
AUTHORS: Juliana Alcoforado Diniz, Dylan Baker, Jessica Garofalo, Paul Robson

[DESCRIPTION]
The dispersed samples were shipped cold from PRODOLABS. Prior to scRNA-seq dispersed samples from brain dead donor’s pancreatic islets were recovered and dissociated as follows.






[STEPS]
SECTION: Abstract
1. The dispersed samples were shipped from PRODOLABS. Prior to scRNA-seq dispersed samples from brain dead donor's pancreatic islets were recovered and dissociated as follows. 
SECTION: Cell Dissociation with Accutase
2. NOTE: Before beginning cell dissociation coat all the materials (pipettes, tubes, etc.) with PIM-S001GMP media to prevent sticking. 
Transfer cell suspension of pure islets to a new 50ml tube. Use additional media to rinse original container.
Centrifuge at room temperature 130g for 5 mins. 
Aspirate the supernatant and add media to the appropriate concentration of 1,000 islets/1 ml.        --> Ex: If receiving 7,000 islets, use 7 ml media.
To recover cells, add half of the islets to a coated flask and incubate at 37C overnight.
Coat pipette and add islets from flask to a 50ml tube. Wash flask with media to make sure all islets have been collected.
Centrifuge at 130 g, room temperature for 2 mins.
Aspirate media and resuspend in 4 ml accutase. Incubate at 37C for 8 mins, mixing with pipette every 2 mins. Check at 6 mins.                                                                                                                                      -->1 mL accutase/1,000 islets
Add CMRL 1066 (Cat. 11530037) media to 25 ml then centrifuge at 230 g.
Aspirate supernatant and resuspend in 1.5 ml of CMRL
Filter through a 40 µm Flowmi.
Count cells using AO/PI (acridine orage/propidium iodide) Cell Viability Kit for Luna-FL automated cell counter.
Proceed to cell fixation.


SECTION: Fixation of Cells &amp; Nuclei for Chromium Fixed RNA Profiling
3. Cells were fixated prior to scRNAseq according to https://dx.doi.org/10.17504/protocols.io.[...] /v1
TITLE: DESEq2 for time series
AUTHORS: Karina Jhingan

[DESCRIPTION]
This is a differential analysis for a time series experiment using DESeq2.

[STEPS]
SECTION: Introduction
1. This protocol is heavily based off of the protocol provided by https://alexslemonade.github.io/refinebio-examples/03-rnaseq/differential-expression_rnaseq_01.html.

Note that DESeq and any other differential analysis requires replicates.
SECTION: Imports/Libraries
2. Citation: “ashr” for LFC shrinkage
Stephens, M. (2016) False discovery rates: a new
deal. Biostatistics, 18:2. https://doi.org/10.1093/biostatistics/kxw041
 

 

SECTION: Data Set up
4. Gene Expression Data Matrix
A csv file where rows are genes and columns are samples/time stamps
 

SECTION: Data Set up
5. Metadata File
A csv file with nrows(metadata) == ncols(datafile)
three columns:
-id: sample name, i.e the column names from the
data file 
-group (i.e time stamp or control group)
-replicate

 

SECTION: Data Set up
6. 
 
You should see TRUE outputted twice 
SECTION: Data Set up
7. Read Data

 

SECTION: Data Set up
8. Set up data matrix

 

SECTION: Data Set up
9. Set up metadata
We are using the naive samples ("0 hours") as a reference/control. 
 

SECTION: Data Set up
10. 
 

 

 

 

SECTION: Data Set up
11. 
 
You should see the following printed out: [1] "reference" "24_hours"  "48_hours"  "72_hours" 
SECTION: DESeq2
13. Create DESeq2 Object
 

SECTION: DESeq2
14. Run DESeq2
 

SECTION: Data Set up
12. 
 

SECTION: DESeq2
15. View Results

 
[1] "Intercept"                        "time_stamp_24_hours_vs_reference" "time_stamp_48_hours_vs_reference"
[4] "time_stamp_72_hours_vs_reference"
 

 

SECTION: DESeq2
16. Normalize results by log fold change
Here we are shrinking log fold change by ashr, for more information on ashr go to  step 2 and view the citation.
 

 

SECTION: DESeq2
17. Set up DESeq results for visualization
Convert the results into a dataframe
 

 

SECTION: Visualizations
18. Plot counts
IL2RA was only chosen as an example, change to your gene of interest into the gene parameter.
 

 

SECTION: Save Results
19. Save DESeq2 results
 

SECTION: Data Set up
3. Set Project Path
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>High-quality, highly enriched mitochondrial DNA (mtDNA) sample enables detection of extremely low-frequency mtDNA variants. When mtDNA is carefully enriched, fragmented in the presence of EDTA and sequenced with unique dual indices by Illumina HiSeq in paired-end mode, it is possible to reliably detect variants at allele frequencies &lt;0.05 %. To be able to detect variants at every position of the mtDNA genome, 'dual' alignment and variant calling strategy is required.</p>

[BEFORE_START]
<p>Ensure that your mtDNA sample used for the sequencing</p>
<p>1) is of high quality and highly enriched from nuclear DNA (nDNA) contamination,</p>
<p>2) 1 mM of EDTA is included into the sonication step of the Illumina library preparation,</p>
<p>3) unique dual indexing is used and reads are sequenced in paired-end mode, and</p>
<p>4) reads are de-multiplexed based on both indices.</p>
<p> </p>
<p>Addition of EDTA (or repair enzymes) has been recommended in order to diminish potential oxidative damage or propagation of other damages during library PCR (Costello et al. 2012, Chen et al. 2017). Whereas dual-indexing significantly reduces the possibility of between-sample cross-contamination (Kircher et al. 2012).</p>
<p> </p>
<p> </p>
<p><strong>References</strong></p>
<p> </p>
<p><strong>Chen, L. et al., 2017.</strong> DNA damage is a pervasive cause of sequencing errors, directly confounding variant identification. <em>Science</em>, 355, pp.752–756.</p>
<p> </p>
<p><strong>Costello, M. et al.</strong>, 2013. Discovery and characterization of artifactual mutations in deep coverage targeted capture sequencing data due to oxidative DNA damage during sample preparation. <em>Nucleic Acids Res.</em>, 41(6), pp.1–12.</p>
<p> </p>
<p><strong>Kircher, M., Sawyer, S. &amp; Meyer, M., 2012.</strong> Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. <em>Nucleic Acids Res.</em>, 40(1), p.e3.</p>

[GUIDELINES]
<p>The unique dual indices require paired-end sequencing and produce R1 and R2 reads. However, to our experience, R2 reads seem to cause more artifactual variant results (mainly GC&gt;TA indicative of oxidative damage). Thus, only use of R1 reads for low-frequency variant detection is recommended.</p>
<p> </p>
<p>If 'Before starting' sample quality requirements cannot be met, more stringent variant calling thresholds - namely allele frequency threshold - may be required and extremely low-frequency variant calling is not possible due to the chemical/biological artifacts present in the sample. Furthermore, it is always advisable to use control samples and confirm the accuracy, preferably in multiple experiments (meaning re-preparation of the libraries from the same case and control samples and sequencing the samples in another sequencing run).</p>

[STEPS]
?. 
?. 
?. 
?. 
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?. 
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?. 
?.
TITLE: Standard Operating Procedure: Vibratomes
AUTHORS: Hong-Yuan  Chu

[DESCRIPTION]
This protocol details the procedure of the standard operating procedure (SOP) describes the safe use of vibratomes in the Chu lab.

[GUIDELINES]
Type of SOP: Hazardous equipment.
Purpose and scope: This Standard Operating Procedure (SOP) describes the safe use of vibratomes in the Chu lab.
Training: All investigators are to receive training and be approved by Dr Chu before using the vibratomes.

[STEPS]
SECTION: Tissue sectioning procedure
1. Remove the blade from manufacturer package and wash it with ethanol and distilled water.
SECTION: Tissue sectioning procedure
2. Install the blade carefully in the vibratome according to the procedure describes in the manual for the specific vibratome used.
SECTION: Tissue sectioning procedure
3. Mount the tissue onto chuck and place in vibratome according to the procedure describes in the manual for the specific vibratome used.
SECTION: Tissue sectioning procedure
4. Submerge the tissue in media appropriate to sectioning purpose (e.g. ice-cold 1x PBS).
SECTION: Tissue sectioning procedure
5. Slice the tissue at desired thickness using the procedure describes in the manual for the specific vibratome used.
SECTION: Tissue sectioning procedure
6. Remove the blade carefully after slicing the tissue and dispose of in a sharps-container.
SECTION: Tissue sectioning procedure
7. Cleanse the vibratome with deionized water.
TITLE: Next of Kin Participation in Care
AUTHORS: Albert Westergren

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Excel database. Next of Kin Participation In care.</span></div><div class = "text-block">Sex (female = 0; male = 1)</div><div class = "text-block">Age (64 years or younger = 0; 65 years or older =1)</div><div class = "text-block">Response to items (disagree (1), somewhat disagree (2), neither agree nor disagree (3), somewhat agree (4), and strongly agree (5))</div><div class = "text-block"><span style = "font-weight:bold;">Excel, raw score to location to metric (score tranformed to interval scores of the same ragne as the original scale)</span></div></div>

[STEPS]
TITLE: Digestion with NEBNext dsDNA Fragmentase (M0348)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
NEBNext dsDNA Fragmentase is an enzyme-based reagent that shears DNA to produce fragments of the desired sizes in a time-dependent manner, for next generation sequencing library preparation protocols
dsDNA Fragmentase provides random fragmentation, similar to mechanical methods (1,2).

[BEFORE_START]
Adequate mixing of NEBNext dsDNA Fragmentase is important for the success of this reaction. NEBNext dsDNA Fragmentase should be vortexed for 3 s prior to use.

For tough digestions, add 1 µL of 200 Milimolar (mM) to the reaction. Additional MgCl2 can be added if necessary.

The protocol listed below is for fragmentation of 5 ng–3 μg of DNA.

[STEPS]
2. Combine the following components in a sterile PCR tube and vortex: 
   component amount   DNA (5 ng–3 μg) 1–16 μl   10X Fragmentase Reaction Buffer v2 2 μl   Sterile Water  variable

3. Add 2 µL and vortex mixture for 3 s.
 

4. Incubate at 37 °C for the recommended times below to generate the desired fragment size:
 

   Desired Fragment Size (bp) Incubation Time (min)   50–200 25–35   200–1,000 15–25   1,000–2,000 10–15   

 

5. Add 5 µL  to stop the reaction.
6. Clean up the fragmented DNA with column purification or using SPRI beads.
 

For further analysis:

Bioanalyzer: Clean up the fragmented DNA prior to loading on a Bioanalyzer chip. 
End Repair: Clean up the fragmented DNA then proceed with desired DNA end repair protocol.
Polyacrylamide Gel Analysis: Clean up the fragmented DNA prior to loading the samples on a PAGE gel.
Long Term Storage: Clean up the fragmented DNA prior to long term storage.
Agarose Gel Size Selection/Analysis: Samples can be loaded directly on to an agarose gel. It is not necessary to clean up the reactions prior to loading.
1. Vortex NEBNext dsDNA Fragmentase for 3 s, quick spin and place on ice.
TITLE: Beetle rearing media
AUTHORS: Yin-Tse  Huang,   Tina

[DESCRIPTION]
ambrosia beetle rearing


[STEPS]
1. The recipe produces enough medium to fill approximately 18 culture tubes.(12cm)
First, add powder or solid
 

3. All ingredients were mixed thoroughly, then put cotton plugs into the mouth of the filled tubes.  

4. The culture tubes was autoclaved at 121 °C for 30 min.

5. Put culture tubes to dry 2 days in oven at 55°C.
(cotton plugs don't remove)
  

2. Second, add liquid
TITLE: EXTRACTS PRODUCTION AND FRACTIONATION
AUTHORS: Rene Flores Clavo, Cristian Daniel Asmat Ortega, Nataly Ruiz Quinones

[STEPS]
?. [Pre-inoculum]
Put three to four colonies of the bacteria  in 5.0 mL of R2A broth supplemented with ASW with NaCl 7%, pH 7.0, and incubated at 28 °C for 5 to 7 days
?. [Inoculum]
After cultures grow, transfer the total volume to an Erlenmeyer of 500.0 mL containing the same culture broth and incubated for 7 days at 28 °C under agitation at 150 RPM followed by 23 days without agitation
?. [Inoculum]
Continue with the bioprocess for 28 days without agitation, maintaining incubation conditions.
?. [Extraction]
Add  Ethyl acetate in culture broth in proportion 1:1 v/v in a beaker of 2 L. Rupture the cells in ultra turrax basic (IKA ref.02H2063.08.CC) at 7000 rpm for 15 min. and leave to macerate overnight.
?. [Extraction]
Obtain the organic fraction and concentrate the crude extract in a round bottom flask of 1L in  the rotary evaporator (R-215 Buchi) under vacuum 200 mbar and temperature of 40 °C until the solvent is completely dried and store it at 4°C
?. [Fractionation]
Fractionate the crude extracts of the isolates using a vacuum C18 chromatographic column for Solid Phase Extraction (SPE).
?. [Fractionation]
Solubilize the initial crude extract in methanol (6,4 mg/mL w/v).
?. [Fractionation]
Obtain the following fractions: 1 water (H2O); fraction 2 water: methanol (H2O: MeOH 1:1 v/v); fraction 3 methanol (MeOH); fraction 4 MeOH: ethyl acetate (EtOAc) (1:1 v/v); fraction 5 EtOAc 100%; fraction 6 EtOAc: Glacial acetic acid 1%
?. [Fractionation]
Dry these fractions in a rotary evaporator at 45 °C under vacuum 200 mbar, weight it and use it for further activities
?. [pre-inoculum]
and incubated at 28 °C for 5 to 7 days
?. [Extraction]
Put the mix in a separatory funnel of 2 L, and recover the organic fraction
?. [Extraction]
Determine the dry weight of the obtained crude extract.
?. [Extraction]
Add again ethyl acetate in the same proportion. Repeat steps 4 and 5 two times, without macerating overnight.
TITLE: Preparation of Bacteria Glycerol Stocks
AUTHORS: Stephane  Fadanka, Shalo  Minette, Nadine             Mowoh

[DESCRIPTION]
This protocol is meant to provide researchers with a step by step procedure on how to prepare glycerol stocks in order to preserve and store bacteria for long term. 
Bacterial glycerol stocks are important for long-term storage of plasmids. The addition of glycerol stabilizes the frozen bacteria, preventing damage to the cell membranes and keeping the cells alive. A glycerol stock of bacteria can be stored stably at -80°C for many years and -20°C for several months.

Glycerol reduces the harmful effects of ice crystals on bacteria which cause dehydration and damage cells through a localised increase in salt concentration leading to the denaturation of proteins. 


[BEFORE_START]
Make sure all materials and reagents needed for this protocol are available

[GUIDELINES]
This protocol describes the steps in preparing bacterial glycerol stocks. This protocol can be performed by anyone with basic molecular biology skills.

[STEPS]
SECTION: Preparing liquid culture of the bacteria to be stored
1. Prepare LB following this protocol depending on the desired amount of LB and subsequent number of glycerol stock tubes needed.

Using an inoculating wire loop, pick up some bacteria colonies from a culture plate and inoculate in an Erlenmeyer flask containing the LB (with the right antibiotic if applicable).

Grow the cells by incubating in an incubator at 37 °C for 180 minto obtain maximum cell growth.

SECTION: Diluting Pure Glycerol to 50% with Distilled water
2. Use a clean measuring cylinder to measure 10 mLof distilled water and equal amount 10 mL of   into a 50 mL falcon tube.

Cork the tube and shake thoroughly until the liquids are evenly mixed

SECTION: Filtering the 50% glycerol
3. Use a 20 mL sterile syringe to aspirate the 50% glycerol from the falcon tube 

Plug in a 0.2 µM micro filter and filter out the glycerol into a sterile falcon tube


SECTION: Aliquoting the bacterial culture into 50% glycerol and storing
4. To make 1mL of Bacteria glycerol stocks, aliquot 500 µLof the filtered 50% glycerol into separate 1.5 mL Eppendorf tubes (triplicates or more depending on the quantity of glycerol stocks needed.
Use sterile micropipette and tips to measure out equal volumes (500 µL) of the bacterial culture from the Erlenmeyer flask into the tubes containing the 50% glycerol (We now have equal proportion of bacteria and 50% glycerol).

Keep your thumb pressed firmly against the lid of the Eppendorf tube and  shake vigorously to make sure the 2 liquids mix completely.

Use a marker pen to label the tubes with the name of the bacterial strain and date of preparation of the glycerol stock

Store the vials in the freezer at -20°C until they are used.
TITLE: Cell subculture
AUTHORS: PMAT0001 

[STEPS]
?. Pre-warm reagents to    in water bath for about .
37 °C
?. Aspirate spent culture media from cell culture vessel.
?. Wash cells once with PBS (  is enough to wash T25 flasks and maybe  for T75 flasks).
2 mL
5 mL
?. Aspirate PBS (from the side of the plate that does not have any cells, so as to avoid disturbing the cells).
?. Add  of Trypsin-EDTA (Add this volume for T25 flasks and accordingly about 5mL for T75 flasks) in the cell culture flasks; Incubate flasks for about .
2 mL
?. After incubation, examine cells under a microscope. Fully trypsinized cells should appear rounded up and no longer attached to the surface of the flask. A few taps can be applied onto the flask to make cells detach faster.
?. Once the cells have detached, add FBS/serum-containing medium to the flask in an equal ratio to the added trypsin. Tyrypsin will start acting on excess serum proteins instead of harming cells.
?. Collect harvested cells and pipette into an appropriate-sized centrifuge tube.
?. Centrifuge cells for   at 1500rpm.
?. After centrifuging, aspirate media and trypsin.
?. Add   of fresh media into the tubes.
2 mL
?. In each of the new flask (T25), add  of fresh media.
5 mL
?. Add about   of cells from step 11 into each of the new flasks.
1 mL
?. [Rule of Thumb for ratio of splitting]
- After the cells are 80% confluent, we should split them as they are in the log phase of growth- Refer to attached images for the rule of splitting
TITLE: Cells electroporation for cell transfection with NEON system
AUTHORS: Juan.Gonzalez

[DESCRIPTION]
Protocol for cell electroporation with the NEON transfection system

[STEPS]
1. Warm the resuspension buffer and the E2 buffer in the water bath out of the water at 37 °C 

2. Put the electrode part inside the left hand side hood in 419
3. Put a NEON glass tube matching the electrode position

4. Add 3 mL of E2 buffer in the glass tube. 
 



5. Switch on the NEON equipment

6. Load the saved settings. 
 


7. Add 5 µg   of the plasmid in a sterile Eppendorf tube
8. Add 3 mL of fresh cell media without antibiotics in a T25 flask


9. Trypsinise the cells as usual
10. Once you have the cell pellet, discard the supernatant carefully
11. Wash the pellet with 150 µL of sterile DPBS and discard the supernatant carefully
13. Resuspend the cell pellet with 130 µL of resuspension buffer and discard the buffer


12. Wash the cell pellet with 150 µL of resuspension buffer and discard the supernatant carefully
14. Transfer 130 µL  of the cell solution to the Eppendorf tube containing the plasmid


15. Mix without making any bubble
17. Take 100 µL of the mixture with the NEON pipette and place it in the glass tube matching the electrode position


18. Press "START" in the NEON display
19. Once the "COMPLETE" message appears in the NEON display, put the cells with the NEON pipette in the T25 flask

20. Put the T25 flask in the “Antibiotics free” incubator


21. After1440 min , check the cells under the microscope and perform the required experiment

16. Take a 100 µL tip with the NEON pipette