eamag/protocols-with-steps huggingface dataset:
TITLE: NEBNext dA-Tailing Module (NEB #E6053)
AUTHORS: New England  Biolabs, Menna Teffera

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The NEBNext dA-Tailing Module has been optimized to efficiently incorporate a non-templated dAMP on the 3 ́ end of a blunt DNA fragment (1). 3 ́-dA DNA tailing prevents concadamer formation during subsequent ligation steps.
DNA tailed with the NEBNext dA-Tailing module may be ligated to adaptors or cloning vectors with complementary dT overhangs. The NEBNext dA-Tailing Module is provided as a master mix to maximize efficiency and convenience in DNA sample preparation workflows.</div><div class = "text-block">
The NEBNext dA-Tailing Module has been validated by sequencing with the Il- lumina Genome Analyzer II (Illumina, Inc.) in conjunction with the NEBNext End Repair Module, NEBNext Quick Ligation Module and Phusion® High-Fidelity PCR Master Mix.</div><div class = "text-block">
For larger volume requirements, customized and bulk packaging is available by purchasing through the OEM/Bulks department at NEB. Please contact OEM@neb.com for further information.</div></div>

[STEPS]
?. [dA Tailing]
Mix the following components in a sterile microfuge tube:  AB1ComponentVolume2End Repaired, Blunt DNAvariable3NEBNext dA-Tailing Reaction Buffer5 µl4Klenow Fragment (3'→5' exo-)3 µl5Sterile watervariable6Total Volume50 µl
AB1ComponentVolume2End Repaired, Blunt DNAvariable3NEBNext dA-Tailing Reaction Buffer5 µl4Klenow Fragment (3'→5' exo-)3 µl5Sterile watervariable6Total Volume50 µl
?. [dA Tailing]
Incubate in a thermal cycler for  at .
37 °C
?. [dA Tailing]
Purify DNA sample on one spin column or using AMPure XP beads.
Note: for details on how this module is used in the NEBNext Library Prep for Illumina workflow, please see the manual for NEBNext DNA library Prep Master Mix Set for Illumina (NEB #E6040).
TITLE: Co-extraction of RNA and DNA from animal tissue
AUTHORS: Dominik Buchner

[DESCRIPTION]
This protocol describes how to co-extract RNA and DNA from animal tissue samples. Samples are homogenized and simultaneously lyzed by bead-beating. Cell debris is pelleted by centrifugation, the DNA is then subsequently bound to a silica column, while the RNA passes the membrane. The RNA in the flow-through is then precipitated with 70% ethanol and bound to a second silica column. Both, DNA and RNA are washed with different wash buffers to remove remaining proteins and other contaminants and finally eluted in separate tubes. If the user is just interested in the RNA, the DNA spin-column can just be discarded.

[BEFORE_START]
Make sure all buffers are prepared before starting.

[GUIDELINES]
Follow general lab etiquette. Wear gloves to prevent contamination of samples. Clean the workspace before starting and after finishing with 80% EtOH.

[STEPS]
SECTION: Sample preparation and lysis
1. For each sample prepare one 2 mL screwcap tube pre-filled with approximately 400 mg of 2 mm zirconia beads and 0.1 mm glass beads.
 

SECTION: Sample preparation and lysis
2. Add up to 30 mg of animal tissue to the prepared tube.

 


SECTION: Sample preparation and lysis
3. Add 1000 µL to the sample tube.

 


SECTION: Sample preparation and lysis
4. Immediately bead beat for 5 min at maximum speed.

 


SECTION: Lysate clearing
5. 10 min 

SECTION: DNA binding
6. Transfer 700 µL of the cleared lysate from step 5the to a silica spin column to bind the DNA in the lysate. Keep the flow-through. Mark the spin column as the DNA column.

 


SECTION: RNA precipitation and binding
7. Add 700 µL to the flow-through from step 6 to adjust the binding conditions for RNA to bind to the silica column.

SECTION: RNA precipitation and binding
8. Vortex the samples to mix the lysate with the ethanol. Do not centrifuge.
SECTION: RNA precipitation and binding
9. Load the mixture on a second spin column. Mark this column as the RNA spin column. 
11000 x g, 15 s  and discard the flow-through.

 


SECTION: Washing steps
10. Add 700 µL to the RNA spin column,11000 x g, 15 s  and discard the flow-through. 
 


SECTION: Washing steps
11. Add 500 µL  to the RNA spin column, add 500 µL to the DNA spin column, 11000 x g, 15 s  and discard the flow-through.

SECTION: Washing steps
12. Add 500 µL  to the RNA spin column, add 500 µL  to the DNA spin column, 11000 x g, 15 s  and discard the flow-through.

SECTION: Column drying and elution
13. 11.000 x g, 1 min to dry the silica membrane of the spin columns. Transfer the spin column to a fresh 1.5 mL microcentrifuge tube.
SECTION: Column drying and elution
14. Add 100 µL directly to the silica membrane. Incubate the column for 3 min at Room temperature
 


SECTION: Column drying and elution
15. 11.000 x g, 1 min , store     the eluted RNA at -80 °C and the eluted DNA at -20 °C
TITLE: 10x Protocols: Visium v1 FFPE Library Construction -- University of Minnesota TMCs (CG000407 Rev C)
AUTHORS: IOx Genomics, Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
Protocols from 10x Genomics for Visium Spatial Gene Expression v1 chemistry on FPPE samples without the CytAssist component. 

Version of protocol used: 
    10x protocol CG000407, Revision C -- Library Construction 

Additional protocols added for guidance:
    10x protocol for imaging guidelines 
    10x protocol for tissue preparation guidelines 

Note: These protocols may not be the current version offered by 10x Genomics but were used to produce the specific datasets connected to them. Please review the 10x Genomics support website for the most recent versions of the protocols prior to starting your experiment. 

[STEPS]
1. 10x protocol CG000407, Rev C (Library construction)
  

2. Additional Protocols/Resources
  
  
https://www.10xgenomics.com/support/spatial-gene-expression-ffpe
TITLE: Spot assay
AUTHORS: Marijn Ceelen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A variant of the plaque assay in which entire phage dilution ranges can be tested on a single plate. </div></div>

[STEPS]
?. Streak LB plate with E. coli (strain should susceptible to phage lambda. Strains that include a prophage lambda are likely to be resistant. Strains LE392 and DH10B have been used with this protocol) and incubate overnight at
37 °C
?. Pick a colony from this plate and use it to inoculate  of LB. Incubate this culture at until OD reaches 2-3. Overnight culture is recommended.
10 ml
37 °C
?. Mix  of cell culture with  of liquid soft LB agar  ().
200 µl
3 ml
50 °C
?. Make a phage dilution range in a 96 wells plate in LB medium. It is advisable to make a wide range of dilutions. 10-1 to 10-12 covers the range of possible titers.
?. Pour the mixture on the preheated LB agar plates and spread the mixture on the plate by moving it. Make sure to work quickly to avoid clumps of solidified agar. Incubate for  at room temperature.
?. Preheat LB agar plates at. Store soft LB agar (LB agar with 0.7% agar) at .
37 °C
50 °C
?. Use a multichannel pipet with 8 channels to gently drop  drops of the phage dilution on the plates. 4 rows of 8 drops fit on a plate. Mark the plate beforehand to indicate the range of dilution. Be careful to not touch the plate with the pipet and to keep the plate steady. Avoid moving the plate to prevent the drops from spilling out.
3 µl
?. Incubate the plates for  at room temperature . Check whether the drops have dried. If the drops are still clearly visible allow the plate to incubate for a longer time.
?. After the drops have dried, turn the plates over and incubate overnight at .
37 °C
?. Count the amount of plaques visible for the individual drops.  Plaque Forming Units (PFU)/mL can be calculated with the following formula: PFU/mL = 𝑁 × 1/𝐷𝐹 × 1/V.N is the number of plaques of lysis counted on the plate (expressed as PFU); DF is the dilution factor and V is the volume of phage dilution pipetted on the plate.
TITLE: Making LB NGM Plates
AUTHORS: Priota  Islam

[DESCRIPTION]
C. elegans is maintained in the laboratory on Nematode Growth Medium (NGM) agar which has been aseptically poured into petri plates. Sometimes they can also be grown on LB-NGM, where the LB in the media will promote better growth of the bacteria the worms feed on. 
The LB-NGM agar medium can be poured into petri plates easily and aseptically using a peristaltic pump. This pump can be adjusted so that a constant amount of LB-NGM agar is dispensed into each petri plate. A constant amount of agar in the plates reduces the need for refocusing the microscope when you switch from one plate to another.

[STEPS]
SECTION: Pre-Autoclave:
1. Book the autoclave (notebook on top of the machine).
SECTION: Pre-Autoclave:
2. Take clean flasks from the glass kitchen (Only the ones with autoclave tape on are sterile)
SECTION: Pre-Autoclave:
3. Measure all the pre-autoclave reagents and add to the flask (Use a new weighing boat and spatula for each reagent. Also, the cholesterol is kept in the fridge.)
SECTION: Pre-Autoclave:
4. Once water is added mix thoroughly and label with autoclave tape (‘LB NGM Rm 5020’). Makesure the bottle is not screwed completely when placing it inside the autoclave machine.
SECTION: Using the Autoclave:
5. Turn ON the autoclave
SECTION: Using the Autoclave:
6.  Make sure that the autoclave’s probe bottle is the same size as the largest bottle you use and fill it with water.
SECTION: Using the Autoclave:
7. Place the temperature probe in it.
SECTION: Using the Autoclave:
8. Fill up the autoclave with water until it reaches the grill.
SECTION: Using the Autoclave:
9. Place the bottles in the autoclave and make sure that the cap is not screwed completely.
SECTION: Using the Autoclave:
10. Check the waste flask is not too full
SECTION: Using the Autoclave:
11. Use ‘media’ program.
SECTION: Using the Autoclave:
12. Press START.
SECTION: Using the Autoclave:
13. It will take about 2.5 hours for 1L or larger bottles.
SECTION: Post-Autoclave:
14. When autoclave is complete, remove the probe flask
SECTION: Post-Autoclave:
15. Make sure to wear gloves as the flask will be hot
SECTION: Post-Autoclave:
16. Let the agar to cool to around 55°C, ie the bottle is cool enough to hold for a second with a gloved hand.
SECTION: Post-Autoclave:
17. Add the post autoclave reagents.
SECTION: Post-Autoclave:
18. Mix it well and start pouring onto desired sized plates (See Protocol for plate pouring)
SECTION: Post-Autoclave:
19. Try not to shake the bottle too much while mixing to avoid air bubbles.
SECTION: Post-Autoclave:
20. The agar needs to be warm to be poured without blocking the tubings, so try to pour as quickly as possible and if not poured immediately put the bottle on a waterbath set to 60C until being used.
TITLE: Plasmid-reprogramming of human fibroblasts
AUTHORS: Pascale Baden, michela.deleidi

[DESCRIPTION]
This protocol details about plasmid-reprogramming of human fibroblasts.

[GUIDELINES]
Adapted from Okita et al PMID: 21460823.

[STEPS]
SECTION: Nucleofection (Day 0)- with Amaxa Nucleofector I/II
1. Prepare nucleofection solution: 
      Human Dermal   Fibroblast Nucleofector solution  82 µL     Supplement  18 µl     Plasmid  10 µg   
 
SECTION: Nucleofection (Day 0)- with Amaxa Nucleofector I/II
2. Nucleofect 700.000 fibroblasts (resuspended in the nucleofection solution) with the P-022 program.
SECTION: Nucleofection (Day 0)- with Amaxa Nucleofector I/II
3. Distribute nucleofected fibroblasts on a 6-Well plate, coated with Matrigel.
SECTION: Nucleofection (Day 0)- with Amaxa Nucleofector I/II
4. Culture cells in DMEM + GlutaMAX supplement (Gibco) + 10 % FBS (Gibco) without P/S.
SECTION: Day 1
5. Change medium to DMEM + GlutaMAX supplement + 10% FBS + 2 1800 FGF2 (Peprotech) with 1% P/S (Millipore).

SECTION: Day 3/4
6. Change medium to E8 medium + 100 micromolar (µM) Sodium Butyrate (Sigma Aldrich 303410-100G) + 0.1 % P/S and change medium every other day.

SECTION: Day 21-28
7. Pick iPS cell colonies.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<span style="color: #333333; font-family: 'Helvetica Neue', Helvetica, Arial, sans-serif; font-size: 14px;">A collection of tutorials for the VIROME application. <br /><br />Wommack, K. E., J. Bhavsar, S. W. Polson, J. Chen, M. Dumas, S. Srinivasiah, M. Furman, S. Jamindar, and D. J. Nasko. 2012. VIROME: a standard operating procedure for analysis of viral metagenome sequences. Standards in Genomic Sciences 6:427-439 [</span><a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3558967/" target="_blank">PMC3558967</a><span style="color: #333333; font-family: 'Helvetica Neue', Helvetica, Arial, sans-serif; font-size: 14px;">] </span>

[STEPS]
TITLE: 城市微生物相調查計畫—採集土壤教學
AUTHORS: Hsin-Mao  Wu

[DESCRIPTION]
吳昕懋

[STEPS]
SECTION: 採土與iNaturalist紀錄教學
1. 移除表土，取距離土表5~7公分之處的土，裝入塑膠夾鏈袋中   
   

SECTION: 採土與iNaturalist紀錄教學
2. 將塑膠袋中的土均勻混合，倒入採集罐之中  
 



SECTION: 採土與iNaturalist紀錄教學
3. 在採集紀錄紙上勾選採樣地點、採樣種類，以及填寫日期、城市、鄉鎮、地點、採集者等資訊
SECTION: 採土與iNaturalist紀錄教學
4. 最後拍兩張照片
 

   
        

 

SECTION: 採土與iNaturalist紀錄教學
5. 接著打開iNaturalist app
SECTION: 採土與iNaturalist紀錄教學
5.1. 如圖所示，點選右下角的加號  

SECTION: 採土與iNaturalist紀錄教學
5.2. 如下圖所示接著需選擇觀察類型，請點「選擇照片」   

SECTION: 採土與iNaturalist紀錄教學
5.3. 請點選左上角紅框處的相機圖示，上傳第二張照片；接著點選圖中央綠框處，定位採集地點  
 

SECTION: 採土與iNaturalist紀錄教學
5.4. 按下打勾處完成定位  

SECTION: 採土與iNaturalist紀錄教學
6. 上傳完成後，若是搭載Android之裝置，可在app中的詮釋資料，找到iNaturalist ID (如下圖紅框處)  



SECTION: 採土與iNaturalist紀錄教學
7. 在採集紀錄紙上填入ID，並將紀錄紙下半部，沿虛線撕下並放入採集罐中    

SECTION: 採土與iNaturalist紀錄教學
8. 記得將採集地點恢復原狀！   

SECTION: 採土與iNaturalist紀錄教學
9. 將採集罐保存於室溫，並在10日內將樣本寄至高雄醫學大學，詳細地址資訊於問卷開頭。

SECTION: 採土與iNaturalist紀錄教學
6.1. 若是搭載iOS之裝置，請在app中的觀察紀錄右上角，點選分享按鈕後，開啟瀏覽器，並在網址列末端找捯iNaturalist ID
TITLE: Modified Dixon’s Agar (Heitman Lab)
AUTHORS: Heitman Lab, Amy Gladfelter

[DESCRIPTION]
Media recipe for growth of Malassezia species.

[STEPS]
1. Malt extract . 36 g

2. Mycological peptone (Oxoid L40)  10 g

3. Dessicated ox bile (Oxoid) . 10 g

4. Agar 20 g

5. Tween 60 (heated before use) 10 mL

6. Glycerol, 50% 4 mL

7. dH2O 986 mL

8. Sterilize by autoclaving at 121 °C for 15 min.


9. Cool to 50 °C.
TITLE: Bleach/Sync Large Scale Culture Plates (LSCP)
AUTHORS: Muhammad Zaka Asif, Man Shah

[DESCRIPTION]
This abstract describes how to bleach/synchronize a large mixed-stage population of C. elegans grown on Large Scale Culture Plates (LSCP). For more information on LSCPs, refer to Shaver et al., 2021. 

[STEPS]
1. Wash LSCP with 50mL M9, Centrifuge and aspirate the supernatant from the wash 15 mL at a time (it should take about 3 centrifuges since some of the buffer will soak into the agar or be irrecoverable).
2. Repeat the 50 mL washes 2 more times for a total of 3 washes. (Shaver et al., 2021)
3. Centrifuge the flip top tubes at 515 RCF for 1 minute. Aspirate out supernatant.
4. Add 2.5 mL of bleach solution (7:1:2 ratio of ddH2O, 5M NaOH, and bleach) to the flip top tube and swirl around the solution. Flip the tubes to ensure the pellet completely dissociates from the bottom and mix vigorously. Vortex if needed.
5. Perform bleaching for only up to 3 tubes at a time to ensure quality.
6. Check the effect of bleaching every 30 seconds by aliquoting a 1 uL sample onto a glass slide to see how the worms are responding.
7. Once most of the adult worms are splitting apart and releasing their eggs, stop the bleach by diluting the tube all the way to 15 mL with M9.
8. Centrifuge immediately at 525 RCF for 1 minute. Aspirate out the bleach solution quickly.
9. Wash egg pellet with 10 mL of M9 once, centrifuging at 309 RCF for 3 minutes. Aspirate out the buffer after each centrifuge. Shake the tubes vigorously after adding the M9 to ensure the pellet dissociates.
10. Add a final volume of 3 mL M9 to the tube and shake just enough to free the pellet from the bottom but without letting the eggs collect on the upper sides of the tube.
11. Transfer to a 50 mL autoclaved flask covered in aluminum foil to minimize cross contamination, and put in a shaker at 1 RCF and 20 C.
12. Allow to grow to L1 arrest overnight.
TITLE: Arabidopsis flower dip transformation
AUTHORS: Magdalena Julkowska

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A standard protocol how to transform Arabidopsis plants using a flower-dip method. 

</div><div class = "text-block">  For higher rates of transformation, plants may be dipped two times at 5-7 day intervals. We suggest one dip two days after clipping, and a second dip one week later. </div></div>

[STEPS]
?. Grow healthy Arabidopsis plants until they are flowering.
?. Prepare Agrobacterium tumefaciens strain carrying gene of interest on a binary vector. Grow a large liquid culture @ 28C in LB with antibiotics to select for the binary plasmid, or grow in other media.
?. Spin down Agrobacterium, resuspend to OD600 = 0.8-1.0 (can be higher or lower) in LB with 5% Sucrose. You will need 250 ml for each four small pots to be dipped.
?. Before dipping, add Silwet L-77 to a concentration of 0.05% (125 ul/250mL) and mix well.
?. Dip above-ground parts of plant in Agrobacterium solution for 10-30 seconds, with gentle agitation. You should then see a film of liquid coating plant.
?. Place dipped plants under a cover in a dark O/N to maintain high humidity (plants should be laid on their side).
?. Water and grow plants normally. Stop watering as seeds become mature.
?. Harvest dry seed. Transformants are usually all independent, but are guaranteed to be independent if they come off of separate plants.
?. Select for transformants using antibiotic or herbicide selectable marker.
?. Transplant transformants to soil.
TITLE: MyHC-IIb/Laminin Fresh Frozen
AUTHORS: Griffen Wakelin

[DESCRIPTION]
Myosin heavy chain IIb/laminin immunofluorescence staining protocol for fresh frozen skeletal muscle.

[STEPS]
SECTION: Staining
1. Thaw sections, dry for ~30 mins, pap, and label.

 

SECTION: Staining
2. Block   60 min Room temperature 

SECTION: Staining
3. Wash 1X PBS 5 min 

SECTION: Staining
4. Wash 1X PBS 5 min 
SECTION: Staining
5. Wash 1X PBS 5 min 
SECTION: Staining
6. Incubate primaries in 1% BSA:
  1:10
  1:1000
  4 °C 

SECTION: Staining
7. Wash 1X PBS 5 min 
SECTION: Staining
8. Wash 1X PBS 5 min 
SECTION: Staining
9. Wash 1X PBS 5 min 
SECTION: Staining
10. Incubate secondaries in 1% BSA:
  1:1000
  1:1000
  1 μg/ml
120 min Room temperature 

SECTION: Staining
11. Wash 1X PBS 5 min 
SECTION: Staining
12. Wash 1X PBS 5 min 
SECTION: Staining
13. Wash 1X PBS 5 min 
SECTION: Staining
14. Mount with aqueous mounting media. Image within 2 days.
TITLE: Mouse Stellate Immunohistochemistry protocol
AUTHORS: Pradeep Rajendran

[STEPS]
?. See mouse steallte isolation protocol
?. Complete/intact stellate ganglia are fixed in 4% paraformaldehyde overnight at 4oC.
?. Fixed tissue is rinsed in phosphate buffered saline (PBS), and stored in PBS + 0.02% sodium azide.
?. The stellate ganglion whole mounts are permeabilized in 'block' solution. (10% normal donkey serum in 1.0% BSA + 0.4% Triton X-100 + PBS).
?. Ganglia are stained with antibodies directed to either tyrosine hydroxylase (TH) or protein gene product 9.5 (PGP 9.5). Secondary staining with streptavidin conjugated to ATTO-647N was used to visualize neurobiotin filling. Stained tissue was rinsed in PBS and mounted on glass slides.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is a two-part lipid extraction for labyrinthulomycetes. The only strain we have tried this protocol on so far is <em>Aurantiochytrium limacinum</em> ATCC MYA-1381.</p>
<p> </p>
<p>This protocol was modified from: Higgins, B.T., A. Thornton-Dunwoody, J.M. Labavitch, J.S. VanderGheynst. 2014. Microplate assay for quantitation of neutral lipids in extracts from microalgae. Anal. Biochem. 465: 81-89. </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is part of the <a href="https://www.protocols.io/view/HERP-Haploid-Engineering-and-Replacement-Protocol-drq55v" target="_blank">HERP protocol</a>, from: <br /> <span class="cit-auth cit-auth-type-author">Alexander WG</span> <span class="cit-sep cit-sep-separator">, </span> <span class="cit-auth cit-auth-type-author">Doering DT</span> <span class="cit-sep cit-sep-separator">,</span> <span class="cit-sep cit-sep-last-separator">and</span> <span class="cit-auth cit-auth-type-author"> Hittinger CT (2014) <a href="http://www.genetics.org/content/198/3/859.full" target="_blank">High-Efficiency Genome Editing and Allele Replacement in Prototrophic and Wild Strains of Saccharomyces</a>. Genetics <span class="cit-vol">198<span class="cit-sep cit-sep-after-article-vol">:</span></span><span class="cit-pages"><span class="cit-first-page">859</span><span class="cit-sep">-</span><span class="cit-last-page">866; </span></span><span class="cit-doi"><span class="cit-sep cit-sep-before-article-doi">doi:</span>10.1534/genetics.114.170118</span></span>

[STEPS]
?. 
?. 
?. 
?.
TITLE: Electrospray ionization analysis of eluted nucleotides
AUTHORS: Annan SI Cook, Xuefeng Ren, Anthony T. Iavarone

[DESCRIPTION]
This protocol describes the denaturation of PI3KC3-C1, and subsequent analysis of eluted nucleotides by electrospray ionization mass spec. 

[STEPS]
SECTION: Buffer Exchange
1. Wash a 5 mL desalting (PD-10) column with 0.5 Molarity (M) NH4CH3CO2.
SECTION: Buffer Exchange
2. Load PI3KC3-C1(VPS15-TSF) and PI3KC3-C1(mCherry-ATG14|VPS15-TSF) protein samples onto a 5-mL PD-10 column.
SECTION: Buffer Exchange
3. Exchange the protein sample buffer by passing the protein through the column.
SECTION: Protein Denaturation
4. Heat the buffer-exchanged protein samples to 90 °C for 10 min to denature the proteins.

SECTION: Centrifugation
5. After denaturation, centrifuge the samples at 21000 x g, 15 min to separate out any precipitated protein.
SECTION: Nucleotide Concentration Assessment
6. Measure the A260 absorbance of the denatured samples using a Nanodrop spectrophotometer.
SECTION: Setup of LC-MS System
7. Connect the LC system (Agilent 1200 series) to the LTQ-Orbitrap-XL mass spectrometer equipped with an ESI source (Thermo Fisher Scientific).
SECTION: LC Column Equilibration
8. Equilibrate the Ultra C18 column with the LC mobile phase solvents according to the manufacturer's instructions.
SECTION: Mobile Phase Preparation
9. Prepare mobile phase solvents A and B:

Solvent A: Water with 10 millimolar (mM) ammonium acetate.
Solvent B: Methanol with 10 millimolar (mM) ammonium acetate.
SECTION: LC Elution Program
10. Set up the LC system with the following gradient program:
SECTION: LC Elution Program
10.1. Isocratic flow at 0.5% (volume/volume) B for 2 min.


SECTION: LC Elution Program
10.2. Linear gradient to 99.5% B over 1 min.
SECTION: LC Elution Program
10.3. Isocratic flow at 99.5% B for 4 min.
SECTION: LC Elution Program
10.4. Linear gradient to 0.5% B over 30 s.
SECTION: LC Elution Program
10.5. Isocratic flow at 0.5% B for 1050 s. 

SECTION: LC Elution Program
11. Maintain a flow rate of 150 µL/min and a column temperature of 40 °C.
SECTION: LC Elution Program
12. Inject 20 µL of the sample into the LC system.

SECTION: Mass Spectrometer Calibration
13. Perform external mass calibration in the positive ion mode using the Pierce LTQ ESI positive ion calibration solution.
SECTION: Data Acquisition
14. Acquire full-scan, high-resolution mass spectra in the positive ion mode over the range of mass-to-charge ratio (m/z) = 300 to 1000 using the Orbitrap mass analyzer.
SECTION: Data Acquisition
15. Set the mass resolution to 60,000 (at m/z = 400, FWHM).
SECTION: Data Analysis
16. Analyze the acquired data using Xcalibur software (version 2.0.7, Thermo Fisher Scientific) for peak identification and interpretation.
TITLE: microfluidic sars cov 2 lamp protocol
AUTHORS: Monica Simion, Alexandru Salceanu

[STEPS]
?. [LAMP Amplification]
LAMP Colorimeteric AmplificationAdd  of Add  ofAdd  ofAdd  of product from step 1Incubate for  at Read the colour of the reaction mix
12.5 µl
2.5 µl
8 µl
2 µl
65 °C
Red - SARS Cov 2 - negativeYellow- SARS Cov2 - positive
?. [LAMP Amplification]
MIX saliva in 1x TE bufferAdd   ofand mix with   saliva sampleIncubate at  for
500 µl
500 µl
65 °C