eamag/protocols-with-steps huggingface dataset:
TITLE: Induction of starvation stress
AUTHORS: Louise Uoselis

[DESCRIPTION]
Induction of starvation stress using EBSS in HeLa cells

[STEPS]
SECTION: Day 1
1. Seed cells, aiming for a confluency of 90 – 100%
at the time of treatment the next day. 

SECTION: Day 2
2. Feed cells for 1 h in an appropriate volume of standard growth media. 
SECTION: Day 2
3. Remove the media, and wash each well 3 times with an ample amount of room temperature PBS (eg. Wash with 3 mL of PBS per well of a 6 well plate). 
SECTION: Day 2
4. After the third wash, add an appropriate volume of Earle’s Balanced Salt Solution (EBSS) to each well/plate. 
SECTION: Day 2
5. After 8 h of incubation in EBSS, harvest all samples for analysis.
TITLE: RNA precipitation 
AUTHORS: Liz Hughes

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol can be used to precipitate RNA to clean it up after extraction or to concentrate it. </div><div class = "text-block">Sodium acetate is highly efficient at precipitating nucleic acids and is the most versatile salt because it does not inhibit many of the reactions that are often performed with purified RNAs. The salt neutralizes the charge on the nucleic acid backbone. This causes the DNA to become less hydrophilic and precipitate out of solution.</div></div>

[STEPS]
?. Make up the volume of your RNA solution to 500μl with RNase free water.
?. Add 50 μl of room temperature 3M Sodium Acetate (pH 5.2-5.5)
?. Add 500 μl of room temperature Propan-2-ol. Mix by inverting a few times.Leave at room temperature for 15-20 mins.
?. Pellet the precipitated RNA by centrifugation at 12,000 g for 15 mins.Take note of the side of the tube where the pellet will be.
?. Note the pellet will appear clear and glassy after the supernatant has been removed. Carefully remove the supernatant and wash the pellet with 500μl 70% ice cold ethanol.Be very carefull not to dislodge the pellet. It will appear white and more visable after the ethanol wash.Spin at 12,000g for 2 mins.Repeat this step one more time. (i.e. 2 ethanol washes)Carefully remove all of the ethanol.
?. Allow the pellet to dry with the tube upside down on tissue at room temperature for 5-30 mins.
?. Resuspend the pellet in RNase free water in an appropriate volume (usually 30-50μl)Assess the RNA quantity and integrity using the NanoDrop and Fragment Analyzer.
TITLE: U Mass - Chronic high-fat feeding
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">A high-fat diet of varying composition and percent fat is administered to induce obesity in mice. High-fat diet induced obesity is causally associated with insulin resistance and type 2 diabetes. </div></div>

[STEPS]
?. High-fat diet is given ad libitum in mice.
?. A stock bag of high-fat diet should be stored in a refrigerator.
?. High-fat diet placed in cages should be replaced 2~3 times a week to avoid spoilage.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes a step-by-step procedure for the production of autologous platelet-rich plasma for topical application in dogs.</p>
<p>This protocol was used in the following publication:</p>
<p>Tambella AM, Attili AR, Dini F, Palumbo Piccionello A, Vullo C, Serri E, Scrollavezza P, Dupré G. Autologous platelet gel to treat chronic decubital ulcers: a randomized, blind controlled clinical trial in dogs. <em>Veterinary Surgery</em>, 43(6), 2014: 726-733. (ISSN: 0161-3499) (DOI: 10.1111/j.1532-950X.2014.12148.x).</p>
<p><a href="http://onlinelibrary.wiley.com/doi/10.1111/j.1532-950X.2014.12148.x/full" target="_blank">http://onlinelibrary.wiley.com/doi/10.1111/j.1532-950X.2014.12148.x/full</a></p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: UC Davis - ß hydroxy butyrate Protocol
AUTHORS: Peter Havel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">When a sample is mixed with R1, AcAc in the sample is broken down to acetone by AADC. Upon addition of R2, 3-HB in the sample is oxidized in the presence of 3-HBDH and Thio-NAD. This oxidation triggers the cyclic reactions. Since the original AcAc in the sample has been removed, only 3-HB is assayed by measureing the rate of Thio-NADH production spectrophotometrically.</div></div>

[STEPS]
?. Reconstitute R1 and R2 using the buffers provided.
?. Add 4 µl of calibrator and sample to each well.
?. Add 270 µl of R1 to each well. Incubate at 37°C for 5 minutes.
?. Add 90 µl of R2 to each well. Incubate at 37°C for 2 minutes. Read at 405 nm. Then continue reading every 30 seconds for 2 minutes.
?. Calculate the slope of the reaction for each well. The assay will be linear so the unknown samples can be calculated as (Sample ΔOD/min ÷ Calibrator ΔOD/min) × Calibrator Concentration.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Please contact Dr. Steven Wilhelm (wilhelm@utk.edu) for additional information regarding this protocol.</p>
<p> </p>
<p>Modified from Wilhelm SW and CG Trick.  1995.  Physiological profiles of <em>Synechococcu</em>s (Cyanophyceae) in iron-limiting continuous cultures. Journal of Phycology, 31:79-85.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: MTT assay
AUTHORS: PMAT0001

[DESCRIPTION]
The MTT assay is based on the conversion of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide into purple formazan crystals by LIVINGcells, which determines the mitochondrial activity of these cells. Viable  cells  contain  NADPH-dependent  oxidoreductase  enzymes  which  reduce  MTT  to formazan.1The  insoluble  formazan  crystals  are  dissolved  using  a  solubilization  solution  (e.g.  acidified isopropanol, DMSO) and the resultant coloured solution is quantified by measuring the absorbance at 500-600nm using a multi-plate spectrophotometer. The darker the solution, the greater the number of viable, metabolically  active  cells2.Since for  most  cell  populations,  the  total mitochondrialactivity  would  be proportional to the number of viablecells, this assay is widely used in characterizingthe cytotoxiceffects of drugs or nanoparticleformulationson immortalized cell line or primary cell cultures. 

[STEPS]
SECTION: Day 0
1. Passage cells which are at 80-90% confluence. Perform the usual trypsinization procedure.
SECTION: Day 0
2. Seed 15,000 cells well (100uL) in complete DMEM (using 1X DMEM) in a 96 well plate. Make sure to fill the peripheral wells with 100uL of water. Only use the inner 60 wells for test wells. Seed cells in one plate solely for the test concentrations (10 concentrations, sextuplicates). Seed another plate just to keep nanoparticle-only, DMEM-only and negative controls.
SECTION: Day 1
3. Remove expired media. 

SECTION: Day 1
4. Without any PBS wash, replace the wells with 100uL of nanoparticle-containing media of different concentrations. Use complete medium for this step (more serum-free or reduced serum media)
SECTION: Day 2
5. Remove ALL media
SECTION: Day 2
6. Replace with 100uL of complete DMEM + MTT (MTT final concentration = 0.5mg/mL in DMEM)
SECTION: Day 2
8. Incubate wells with MTT for 120 min  at 37 °C 

SECTION: Day 2
9. Remove all media + MTT from wells

SECTION: Day 2
10. Add 100uL DMSO into all the wells and shake for 10 s at 70rpm on an orbital shaker.

SECTION: Day 2
12. Place the plates in a microplane reader. Shake the plate using the in-built shaker feature for at least 45 s at 425 cpm (fast) using double orbital shaking method. Following that, take absorbance measurements of wells @ 570nm using a microplane reader

SECTION: Day 2
7. After adding DMEM +MTT, shake the plates for 30 s at 70rpm on an orbital shaker.

SECTION: Day 2
11. Incubate for 15 min at 37 °C
TITLE: RT-PCR amplification for the discrimination between wild type and mutation alleles in SARS-CoV-2 genomes from wastewater
AUTHORS: Ana C Reis, Daniela Pinto, Mónica V. Cunha

[DESCRIPTION]
This protocol describes the procedure of RT-PCR for the allelic discrimination between wild type and mutation alleles in specific positions of the SARS-CoV-2 genome. Starting with isolated RNA from SARS-CoV-2 positive wastewater samples, this workflow combines Applied Biosystems TaqMan SNP Genotyping Assays with a one-step RT-PCR reaction to detect whether there are known SARS-CoV-2 mutations present in the wastewater samples.

Sequence-specific forward and reverse primers amplify the target sequence region in each mutation assay. The reverse primer also primes reverse transcription of the SARS-CoV-2 genomic RNA sequences. Each mutation assay includes two TaqMan minor groove binder probes with nonfluorescent quenchers: one VIC dye labelled probe to detect the reference sequence and one FAM dye labelled probe to detect the mutation sequence.

[BEFORE_START]
Wipe bench surfaces with RNAse Away and keep working environment clean. Maintain the RNA samples on ice, and prepare the RT-PCR mixture on ice. 

[STEPS]
SECTION: RT-PCR amplification
1. Prepare the amplification mixture as follows:

 
To the 15 μl of reaction mix, add 5 μl of wastewater RNA sample, or no-template control (DNAse/RNAse free water), or a wild-type AcroMetrix Coronavirus 2019 (COVID-19) RNA control, to complete 20 μl of reaction volume.
2. Thermocycling conditions

 
Set the detection module for FAM and VIC labelled probes.
SECTION: Data analysis
3. The allelic discrimination results can be graphed on a scatter plot contrasting reporter dye florescence (i.e., allele X versus allele Y), and analyzed with the QuantStudio Design and Analysis Software, using the genotyping analysis module with real-time data.
TITLE: Typing E. coli ATCC 11303 with fluorescently stained virus.
AUTHORS: Dr. Steven Wilhelm

[DESCRIPTION]
<p>Please contact Dr. Steven Wilhelm (wilhelm@utk.edu) with any additional information regarding this protocol.</p>

[STEPS]
?. Grow a 4 hour culture of ATCC 11303 in Fe-free LB by inoculating ATCC 11303 from an overnight culture grown in Fe-Free media into fresh media.
[ATCC 11303 culture]
?. Add 4 hour ATCC 11303 culture into a sterile 1.5 mL tube.
[of 4 hour ATCC 11303 culture]
?. Add YO-PRO stained T5.
[YO-PRO]
add 10 ul DAPI, if needed.
?. Allow culture to sit in the dark at room temperature.
?. Add 500 μl of 10 mM MgSO4.
[of 10 mM]
?. In centrifuge, spin cells down at ~ 10,000 rpm, for 3 minutes.
[at 10,000 rpm]
?. Gently remove the supernatant with a pipette, then re-suspend the bacterial pellet in 1.5 ml of 10 mM MgSO4.
[of 10 mM]
?. Filter the sample onto 0.2 μm pore-size, black polycarbonate filter.
?. Rinse the filter gently, three times, with 1 ml of 10 mM MgSO4.
[of 10 mM]
?. Finally, with a clean slide and cover glass, pipet a drop of glycerol or immersion oil in the middle of the glass slide and place the cover glass on top of it. Let the glycerol spread out evenly under the cover glass, removing any bubbles. Remove the cover slip from the slide, place the filter on the slide and replace the cover slip.
TITLE: Overnight culture in Round-bottomed tubes
AUTHORS: Wolfram Moebius

[DESCRIPTION]
This protocol describes how to produce an overnight bacterial culture. 

[STEPS]
SECTION: Prepare overnight culture
1. Create a streak plate and incubate at 37 °C   . 

SECTION: Prepare overnight culture
2. Label a 14 mL round-bottom falcon tube with name, date, strain and any other contents. Transfer 5 mL LB media into the falcon tube. 


 

SECTION: Prepare overnight culture
3. Using an inoculation loop, pick an E.coli isolated colony from the middle of the streak plate and suspend in the 5 mL LB media. 

SECTION: Prepare overnight culture
4. Incubate the inoculated LB media   at 37 °C, shaking at  .
TITLE: Case - Intraperitoneal Insulin Tolerance Test
AUTHORS: Henri Brunengraber

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This is the standard protocol for routine insulin tolerance testing. It is performed on awake mice, fasted for 18 hr (overnight) or for 6 hours.</div></div>

[STEPS]
?. Fast mice for overnight or for 6 hours. Remove mouse from cage and put into a clean cage with water and no food.
?. Label the mice using a marker on their tail to quickly tell them apart
?. Weigh each mouse and record weight. Calculate the amount of insulin needed for 0.75U to 1.0 U /kg body weight. Record the volume for each mouse.
?. Dilute the stock of insulin (100U/ml) to 0.1u/ml
?. Take a glucose reading and record it as time zero. Dip tail into clotisol to clot blood.
?. Inject each mouse 3’ apart. Repeat #4 at 15min, 30 min, 60 min after IP injection of insulin.
?. Return mice to cage with food.
TITLE: No Title
AUTHORS: 

[BEFORE_START]
Make sure you have all necessary equipment, reagents and PCR primers before begining the protocol.

For preparation of multiple samples please, use 'START EXPERIMENT' then 'SCALE PROTOCOL'.

[GUIDELINES]
Workflow was designed and optimised for sample preparation of 16S rRNA gene amplicons with the use of random hexamer-free Rolling Circle Amplification followed by MinION MK1b sequencing.

However, this protocol can be customised for amplification of any prokaryotic and eukaryotic gene from ~200 to ~2000bp.


[STEPS]
SECTION: PCR amplification of 16S rRNA gene
?. 
SECTION: PCR product clean up
?. 
SECTION: PCR product concentration estimation
?. 
SECTION: Self-ligation for the formation of plasmid-like structure
?. 
SECTION: Reverse phase cleanup
?. 
SECTION: Plasmid and short amplicon clean-up
?. 
SECTION: Rolling Circle Amplification (RCA)
?. 
SECTION: First step enzymatic de-branching
?. 
SECTION: Mechanical fragmentation
?. 
SECTION: Secondary enzymatic de-branching
?. 
SECTION: Cleanup of post fragmented RCA products
?. 
SECTION: Gap-filling and dA-tailing of fragmented RCA products
?. 
SECTION: Library preparation and nanopore sequencing (Fig.1I)
?. 
SECTION: Removal of linear molecules from plasmid mix
?. 
SECTION: Cleanup of fragmented RCA products
?. 
SECTION: Cleanup of end-repaired and dA-tailed RCA products
?.
TITLE: CAMbank: cfDNA BCT Field Processing v1
AUTHORS: Eliah G Overbey, Krista A Ryon, jak, Christopher E Mason

[DESCRIPTION]
Field processing of cfDNA BCTs for the Cornell Aerospace Medicine Biobank (CAMbank).

Instructions for preserving: plasma and RBC Pellets.

[STEPS]
SECTION: Perform Venipuncture
1. After venipuncture, invert the tubes gently 8 to 10 times to fully mix tube anticoagulant with blood sample.

Store the tube upright at room temperature until centrifugation.

Note: The tube stabilizes cell-free DNA for up to 14 days at 6 °C to 37 °C and CTCs for up to 7 days at 15 °C to 30 °C.
SECTION: Centrifuge Settings: Plasma Separation
2. Note: A swing bucket centrifuge is required. 

Set centrifuge:
acceleration: 9
deceleration: 9
temperature: RT
duration: 20 minutes
speed: 300xg
SECTION: Centrifuge Settings: Plasma Separation
3. Place the cfDNA BCTs in the centrifuge. 
Place a protective cover over the swing buckets in case of tube leakage. 
Start the centrifuge. 
Stand by the centrifuge until the centrifuge reaches max speed. Listen for signs of imbalance or compromised tube integrity. 
SECTION: Separate Plasma
4. Carefully remove cfDNA BCTs from the centrifuge and inspect for separation of red blood vs plasma layers. 

Transfer cfDNA BCTs to a sanitized laminar flow hood. 
SECTION: Separate Plasma
5. Remove the upper plasma layer and transfer to a new 15mL conical tube. 
Cap the conical tubes and return to the centrifuge. 
SECTION: Aliquot Plasma
9. Carefully remove conical tubes from centrifuge. 
Transfer conical tubes to a sanitized laminar flow hood. 
SECTION: Aliquot Plasma
10. Using a P1000, carefully aliquot plasma into 2D barcoded tubes at 500µL each. 

Return 2D tubes with plasma to the rack and place in the -80C freezer.
SECTION: Centrifuge Settings: Plasma Purification
6. Note: A swing bucket centrifuge is required. 

Set centrifuge:
acceleration: 9
deceleration: 9
temperature: RT
duration: 10 minutes
speed: 5000xg
SECTION: Centrifuge Settings: Plasma Purification
7. Place the conical tubes in the centrifuge. 
Place a protective cover over the swing buckets in case of tube leakage. 
Start the centrifuge. 
Stand by the centrifuge until the centrifuge reaches max speed. Listen for signs of imbalance or compromised tube integrity. 
SECTION: Freeze Red Blood Cell Pellets
8. Recap and freeze the cfDNA tubes to preserve DNA in the red blood cell pellet
TITLE: Total nucleic acid extraction - NucleoMag® DNA/RNA Water Kit (MACHEREY-NAGEL Inc.)
AUTHORS: Adélaïde Roguet, Melissa Schussman

[DESCRIPTION]
Total nucleic acid extraction from wastewater using NucleoMag® DNA/RNA Water Kit (Catalog no. 744220.1, MACHEREY-NAGEL Inc., Duren, Germany).

[BEFORE_START]
1. Clean the working area and all equipment: wipe down with 10% bleach and let dry. Wipe down with 70% ethanol and let dry. Then, wipe down using RNase AWAY and let dry. 

2. Prepare the 6 purification plates: 
    - Sample plate: Add 400μL lysate, 25 μL NucleoMag®B-Beads (vortex the bottle at max speed before use), and 475 μL MWA2 into each well required for purification.
    - Wash 1 plate: 850 μL MWA3 to each well required for purification.
    - Wash 2 plate: (same as plate Wash 1): 850 μL MWA3 to each well required for purification.
    - Wash 3 plate: Add 850 μL MWA4 to each well required for purification.
    - Elution plate: Add 100 μL RNase-free H2O to each well required for purification.
    - Tip plate: Place KingFisher 96-tip comb into an empty KingFisher 96-well plate. While opening the 96-tip comb plate, pay attention to not touch the tips.




[GUIDELINES]
When work is completed, remove equipment and supplies from the cabinet. Wipe the work area with 10% bleach, let stand for 10 min, rinse with water, then with 70% ethanol, and finally with RNAase AWAY.

[STEPS]
SECTION: Total nucleic acid extraction
1. For HA filter extraction, let the sample thaw on ice and go to step 2. 

For BCoV/BRSV extraction (in duplicate), add 5 µL of BCoV/BRSV solution to the 2-mL tube containing 475 µL MWA1. Vortex for 15 seconds (speed 7 out of 10) and flash freeze the tube. Go to step 4.

For Direct extraction, add 150 µL of wastewater to the 2-mL tube containing 100 µL CTAB. Vortex for 15 seconds (speed 7 out of 10) and flash freeze the tube. Go to step 4.
SECTION: Total nucleic acid extraction
2. 

For HA filter extraction, place the 2-mL tubes in the bead beater.
 

SECTION: Total nucleic acid extraction
2.1. Bead beat for 150 s 
 


SECTION: Total nucleic acid extraction
2.2.  Cooldown the samples on ice for 5 min .

SECTION: Total nucleic acid extraction
2.3. Repeat Steps 9.1 and 9.2 once  .

SECTION: Total nucleic acid extraction
3. Centrifuge at maximum speed for 1 min at room temperature. 150000 rpm, 1 min 

SECTION: Total nucleic acid extraction
4. For HA filter extraction, transfer 400 µL of supernatant to the Lysis/Bind plate. 
For BCoV/BRSV/Direct extraction, transfer all supernatant to the Lysis/Bind plate.


SECTION: Total nucleic acid extraction
5. Start the protocol MN_96_Flex.bdz on the KingFisher Flex  74 min 

 


SECTION: Total nucleic acid extraction
6. Transfer the purified sample from the Elution plate to the microcentrifuge tubes.

 

SECTION: RT-ddPCR
7. Quantification by Droplet Digital PCR (ddPCR)

dx.doi.org/10.17504/protocols.io.bpg6mjze
TITLE: Identification of the different phylogroups from the Klebsiella pneumoniae species complex by real-time PCR
AUTHORS: Elodie  Barbier, Carla  Rodrigues, Sylvain  Brisse, Pascal  Piveteau

[DESCRIPTION]
  KpSC phylogroup ID

We developed and optimized a real-time PCR for the identification of the different phylogroups (Kp1 to Kp6) belonging to Klebsiella pneumoniae species complex (KpSC) by amplification of five specific gene targets. 

[STEPS]
SECTION: Primers and Probes
1. 
The KpSC phylogroup ID : Primers, Probes and Amplicon length

  
     PhG -Target      Forward (5‘ – 3‘)      Reverse (5‘ – 3‘)      Probe      Quencher      Reporter      Amplicon length (bp)        Kp1 - group_8970      CCTCAAACACGCCAATATGC      TACCGCGACGAGTAAAGTGG      GATCCATTGATTCCATTCGAACCGG      BHQ-1      JOE      85        Kp2 - group_10724      TTGTTGATTGGCAGGCCTTC      AAATGCTGTGACCACCGTTG      CGCCTGGCGTCAGTGGCCCCGAGC      BHQ-1      JOE      70        Kp3/Kp5 - tetR      GCGGGCCGGCTTTTC      CGCATCCCAGGGTATATTCG      AGGCCGAAGCCGCAATGATATTACTCA      BHQ-1      FAM      71        Kp4 - nanK      AAACAGCGGATGCTCTGAAC      ATGAGTCCGCCAAGATTCTG      ATAGCCTCGACCGGGATTATTCGTGA      BHQ-2      TAMRA      145        Kp6 - mdtG_1      GCGTTTCAATGATCCTGTCC      ATCTGCGTCTGGAAAACAG      GTGCGCTGATTGGTATTGGATATGGC      BHQ-2      TAMRA      76   
 
SECTION: PCR mix preparation
2. PCR mix preparation for each set of primers and probe
 
     Mix Reagents      Volume per   well (𝛍L)        TakyonTM Rox PROBE   MasterMix 2x      12.5        Forward (500 nM)      1        Reverse (500 nM)      1        Probe (250 nM)      0.5        DNA template       2.5        PCR grade water      2.5   
 
SECTION: Amplification program
3. 
 
            Temperature (°C)      Time (minutes:seconds)        Holding stage      95.0      03:00        Cycling stage (x40)      95.0      00:15        60.0      01:00   
 
SECTION: Positive and negative controls
4. 
Negative control: PCR grade water
Positive controls: 
Kp1 DNA (Klebsiella pneumoniae ATCC 13883T) at concentration 2 ng/μl ; 2.5 μl used i.e., 5 ng. 
Kp2 DNA (Klebsiella quasipneumoniae subsp. quasipneumoniae 01A030T)
Kp3 or Kp5 DNA (Klebsiella variicola subsp. variicola F2R9T; Klebsiella variicola subsp.tropica 1266T)
Kp4 DNA (Klebsiella pneumoniae07A044T)
Kp6 DNA (Klebsiella quasivariicola KPN1705T)
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol, developed in 2011 by Angela Poole while in the Weis lab, is a quick prep for protein extraction from Aiptasia adults.</p>

[BEFORE_START]
<p><strong>RIPA extraction buffer:</strong></p>
<ul>
<li>100mM Tris, pH 7.4</li>
<li>100mM NaCl</li>
<li>10mM EDTA, pH 8.0</li>
</ul>
<p> </p>
<p><span style="font-weight: 400;">*Bring to desired volume with Nanopure water and just before use, dissolve </span> <span style="font-weight: 400;">one cOmplete™, mini, protease inhibitor tablet in 10mL of RIPA Buffer. </span><span style="font-weight: 400;">Be sure to let the tablet dissolve on its own- do not vortex!!</span></p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Long-Term Storage of Bacteria
AUTHORS: Huan Jui Chang, Hung Liang Pai

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is suitable for preparing bacterial storage for -80 degree Celsius with glyceral as the solvent. It is usually performed after the overnight incubation of transformed bacteria that are ready for plasmid extraction. </div></div>

[STEPS]
?. [Preparation]
Turn on the UV light of the laminar flow for  to   in order to sterilize it.
?. [Protocol]
Add 300 μL of 50% glycerol into an eppendorf.
?. [Protocol]
Add 600 μL of bacterial media prepared in a procedure like "Bacterial Culture for Plasmid Extraction" to an eppendorf.
?. [Protocol]
Store the eppendorf under -80 degree Celsius.
TITLE: Protocol for variant calling in SARS-Cov-2 enabling long indel detection
AUTHORS: Juanjo Bermúdez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for SARS-Cov-2 variant calling departing from raw reads in a run. First, we will de-novo assemble the genome using s-aligner, then we will use standard software for variant calling: BWA, samtools, and freebayes.</div><div class = "text-block">The advantage of this protocol is that it also finds long indels if they exist. Other protocols based on reference mapping will often miss these.</div></div>

[STEPS]
?. Assemble the genome
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?. Call variants.sh to find variants. The script will call BWA, samtools, varscan and freebayes to generate a VCF file with the resulting variants.