eamag/protocols-with-steps huggingface dataset:
TITLE: READDI protocol: Crystallisation of CHIKV nsP3 macrodomain
AUTHORS: Jasmin Aschenbrenner, Peter Marples, michael fairhead, Andre Schutzer de Godoy, Daren Fearon

[DESCRIPTION]
Chikungunya virus (CHIKV) causes severe fever, rash and debilitating joint pain that can last for months or even years. Millions of people have been infected with CHIKV, mostly in low- and middle-income countries, and the virus continues to spread into new areas due to the geographical expansion of its mosquito hosts. The crystallization protocol and buffer conditions used to obtain reproducible Chikungunya Virus nsP3 macrodomain crystals suitable for XChem fragment screening.

[GUIDELINES]
N/A

[STEPS]
SECTION: Crystallization experiment
3. Protein and buffer requirements:
21.6 µL11 mg/mL  
2.88 mL 
10.08 µL   Seeds, dilution 1:100

SECTION: Crystallization experiment
5. Dispense 30 µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 75 nL11 mg/mL    to each lens using the SPT mosquito.
Dispense  40 nL to each lens using the SPT mosquito.
Dispense  35 nL to each lens using the SPT mosquito.

Drop ratio: 15:8:7 ratio (75 nl   : 40 nl Crystallisation solution: 35 nl Seeds)
Final drop volume: 150 nl
SECTION: Crystallization experiment
4. Crystallisation screen composition:
0.1 Molarity (M)  Tris-NaOH pH 7.8  
0.1 Molarity (M)  Potassium thiocyanate
0.1 Molarity (M) Sodium bromide
25 % v/v PEG Smear Broad

Stock solutions used:
1 Molarity (M) Tris adjusted to pH 7.8 with NaOH
1 Molarity (M)  Potassium thiocyanate
1 Molarity (M)  Sodium bromide
50% v/v PEG Smear Broad

 

SECTION: Crystallization experiment
7. 
 


SECTION: Crystallization experiment
6. Incubate at 20 °C for 1440 min in Formulatrix Rock Imager.

Imaging Schedule: The first images are taken after 12 h and the imaging schedule follows a Fibonacci sequence of days for further collections. 
SECTION: Equipment needed
1. Formulatrix Rock Imager (or incubator of choice)
SPT mosquito 
 
P100 8 multi-channel pipette
SwissCI 3 lens plate

SECTION: Crystallization experiment
2. Prepare seed stock:
 
1: 100 dilution    seeds
SECTION: Data collection at Synchrotron
8. Diamond Light Source
Unattended Data Collection (UDC)
Data Collection Temperature: 100K
Detector: DECTRIS EIGER2 X 9M
Beamline: I04-1
Wavelength:  0.9212 Å
Resolution (Å): 1.64
Beam Size (μm): 60 X 50
Number of images: 3600
Oscillation: 0.10°
Exposure (s): 0.0020
Transmission (%): 100
Flux (ph/s): 3.80e+12
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Cas9 Nuclease, <em>S. pyogenes</em>, (Cas9) is a double-stranded DNA endonuclease that is guided to its target by sequence complementarity of a small RNA loaded into the protein. This protocol describes how to digest double-stranded DNA <em>in vitro</em> using Cas9 and a single guide RNA (sgRNA). </p>

[BEFORE_START]
<p> </p>
<p>Prepare 300 nM sgRNA by diluting the stock with nuclease-free water on ice.</p>
<p>Prepare 30 nM substrate DNA with a single target sequence by diluting the stock with nuclease-free water on ice.</p>

[GUIDELINES]
<p>It is essential to keep the molar ratio of Cas9 and sgRNA per target site at 10:10:1</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: JAX-Sen: Collection and shipment of specimen for Visium Spatial Transcriptomics (vST/Visium ST)
AUTHORS: Laura Robinson, Susan Sheehan, Gaven Garland, Ron Korstanje

[DESCRIPTION]
These samples are part of the JAX-Sen project in the SenNet Consortium. Here we provide details on specimen collection and shipment procedures for Visium Spatial Transcriptomics (vST/Visium ST).

[STEPS]
SECTION: Reagents and Materials:
1. 10% NBF fixative
Tweezers
Appropriate container for fixing
SECTION: Quality Key Points:
2. The tissue specimen should be always kept at 4 degrees Celsius and RNase-free. 
It is crucial to not store the tissue specimen at RT to avoid any cell death, and tissue and/or RNA degradation. 
SECTION: Procedure:
3. Collection/Harvest:
SECTION: Procedure:
3.1. Animal was euthanized via cervical dislocation. 

SECTION: Procedure:
3.2. Animal was pinned to a necropsy tray which remained on wet ice throughout the harvest. 
SECTION: Procedure:
3.3. Animal was perfused with 20ml cold PBS.
SECTION: Procedure:
3.4. Heart, pancreas, and kidney was collected and the specimen was fixed in 10-20 X volume of 10% NBF fixative for 24-48 hours at room temperature.
SECTION: Procedure:
3.5. Samples were kept at 4 degrees Celsius until submission to histology for processing and embedding.
TITLE: 10Xv2 RNASeq Sample Processing
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Allows for rapid generation of 3’ transcriptomic-NGS-ready- single-cell-libraries from pools of cells.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: 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.</span></div></div>

[STEPS]
TITLE: Preparation of unilamellar liposomes
AUTHORS: Ayan Adhikari, Suzanne R Pfeffer

[DESCRIPTION]
We present here a protocol for preparing liposomes that can be used to monitor binding of proteins to vesicles of various membrane curvatures.  We used this to study the association of the PPM1H phosphatase with highly curved membranes due to its N-terminal amphipathic helix.

[STEPS]
SECTION: Make lipid stocks
1. All  lipids were purchased from Avanti Polar Lipids. PC and PS were obtained as 10mg/ml
chloroform solutions. PI, PI(4)P and cholesterol sulfate were obtained as powders. Dissolve in chloroform to prepare 2mg/ml, 1mg/ml and 25mg/ml stock solutions respectively.
SECTION: Prepare the lipid mixture
2. Prepare unilamellar vesicles by mixing:

940 µL of (18:0-20:4)PC
470 µL of (18:0-20:4)PI
60 µL of (18:0-18:2)PS
140 µL of (18:1)PI(4)P
26 µL of cholesterol sulfate 

from the respective stock solutions in a glass vial. The lipid composition of the liposomes is in the ratio (mol %) 78:7:5:1:9 to represent the mammalian cell Golgi composition (Fasimoye et al., 2023).
SECTION: Prepare the lipid mixture
3. Dry the above-mentioned lipid mixture in chloroform under nitrogen flow by pointing a Pasteur pipette into the glass vial. This vial is subsequently incubated under house vacuum for at least 60 min.

SECTION: Prepare the lipid mixture
4. Resuspend the dried lipids by pipetting up and down in 1ml of resuspension buffer (50mM HEPES pH 7.5, 120mM KCl)
SECTION: Liposome preparation
5. Sonicate the liposome suspension by two brief 5 s cycles of bath sonication followed by sequential extrusion through 0.4, 0.1 and 0.05 µm pore size polycarbonate filters for 21 times using a hand extruder.

SECTION: Liposome storage
6. The final lipid concentration of the liposome suspension will be 15mM;  store at 4 °C for a maximum of 2 weeks.  Small liposomes are best used close to the time of their preparation.
TITLE: Purification of CCPG1-GST
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol details the purification of CCPG1-GST, its analysis by SDS-PAGE and Coomassie staining.

[STEPS]
SECTION: Purification procedure
1. To purify CCPG1-GST, fuse the cytosol-exposed domain of CCPG1 (1-212aa) to a C-terminal GST-tag through gene synthesis and Gibson cloning into a pET-DUET1 vector. 

SECTION: Purification procedure
4. Collect the cells by centrifugation and resuspend in lysis buffer. 

Lysis Buffer:

 
 
SECTION: Purification procedure
3. Once the cells reach 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 procedure
2. After the transformation of the pET-DUET1 vector encoding CCPG1-GST in E. coli Rosetta pLysS cells (Novagen Cat# 70956-4), grow 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 procedure
10. Pool the supernatant fractions, filter through a 0.45 µm syringe filter, concentrate with 30 kDa cut-off Amicon filter (Merck Millipore), and load onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva). 

SECTION: Purification procedure
9. To collect the supernatant, collect the beads by centrifugation. Wash the beads twice with 4 mL of wash buffer, and collect the supernatant each time. 

SECTION: Purification procedure
8. Incubate the beads 120 min with 4 mL of 50 millimolar (mM) reduced glutathione dissolved in wash buffer at 4 °C, to elute CCPG1-GST from the beads. 

Wash Buffer: 

 
 
SECTION: Purification procedure
7. Centrifuge the samples to pellet the beads and remove the unbound lysate. Then wash the beads twice with wash buffer, once with high salt wash buffer, and two more times with wash buffer.

Wash Buffer:

 
 
Salt wash Buffer:

 
 
SECTION: Purification procedure
6. 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 CCPG1-GST. 

SECTION: Purification procedure
5. Sonicate the cell lysates twice for 30 s and clear by centrifugation at 18000 rpm, 45 min, 4 °C in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific). 

SECTION: Purification procedure
13. After concentrating the purified protein, aliquot the protein and snap-frozen in liquid nitrogen.

SECTION: Purification procedure
12. Analyze the fractions by SDS-PAGE and Coomassie staining. Pool the fractions containing purified CCPG1-GST. 

SECTION: Purification procedure
11. Elute the proteins with SEC buffer. 

SEC Buffer:
TITLE: LRRK2:DARPins complex preparation
AUTHORS: Marta Sanz Murillo

[DESCRIPTION]
Protocol used to create LRRK2-RCKW: DARPin complex for cryo-EM grid preparation.







[STEPS]
SECTION: LRRK2:DARPins complex preparation
1. 
His6-Z-TEV-LRRK2-RCKW was expressed and purified as described in a previous protocol 


 

SECTION: LRRK2:DARPins complex preparation
2. Prepare LRRK2 buffer exchange: Keep it at 4ºC.
20 millimolar (mM) HEPES pH=7.4
150 millimolar (mM) NaCl
2.5 millimolar (mM) MgCl2
20 micromolar (µM) GDP
0.5 millimolar (mM) TCEP
SECTION: LRRK2:DARPins complex preparation
3.  
Spin down purified LRRK2-RCKW (10000 rcf, 4°C, 10 minutes). Leave protein on ice afterward.



 


SECTION: LRRK2:DARPins complex preparation
4. Exchange buffer using a spin desalting column (Zeba™ Spin Desalting Columns, 7K MWCO (Catalog number: 89877).  
SECTION: LRRK2:DARPins complex preparation
5. 
Spin down again the exchange buffer LRRK2-RCKW (10000 rcf, 4°C, 10 minutes) and measure the concentration. Leave protein on ice afterward



SECTION: LRRK2:DARPins complex preparation
6. Thaw the DARPin protein of your interest (E11 or C12) and spin it down. Measure its concentration.



SECTION: LRRK2:DARPins complex preparation
7. Based on LRRK2-RCKW concentration, add the necessary volume to get a proportional ratio
LRRK2:DARPin 1:1.25 and dilute to a final 10 micromolar (µM) LRRK2-RCKW concentration using exchange buffer (150 mM NaCl)

SECTION: LRRK2:DARPins complex preparation
8. Incubate 10 minutes at RT. Afterward, keep it on ice until grid preparation.
TITLE: 2. Data collection protocols
AUTHORS: Haydeh Payami

[DESCRIPTION]
This is a collection of protocols for data and specimen collection, designed for self-administration by subject. It includes the Environmental & Family History Questionnaire, the Gut Microbiome Questionnaire, instructions for saliva collection, and instructions for stool collection at home.

[STEPS]
SECTION: Data collection protocols
1. 
 

SECTION: Data collection protocols
2. 
 

SECTION: Data collection protocols
3. 
 

SECTION: Data collection protocols
4.
TITLE: SocialInclusionIOS
AUTHORS: j.vyrastekova 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Online questionnaire to assess social inclusion of the SEN students as reported by their parents/care-takers, see attached:</div></div>

[STEPS]
?. Collect data from parents of special education needs students (SEN) age 4-12, by distributing the online questionnaire among parents/care-takers of the SEN students.
TITLE: Quick Protocol for Oligonucleotide Cleanup Using the Monarch® PCR &amp; DNA Cleanup Kit (5 μg) (NEB #T1030)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
Quick Protocol for Oligonucleotide Cleanup Using the Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030)

[BEFORE_START]
All centrifugation steps should be carried out at 16000 x g (~13000 rpm).
Add isopropanol to Monarch DNA Cleanup Binding Buffer prior to use*:

For the 50-prep kit, add 14 mL of isopropanol to the DNA Cleanup Binding Buffer.
For the 250-prep kit, add 63.5 mL of isopropanol to the DNA Cleanup Binding Buffer.

Add ethanol to Monarch DNA Wash Buffer prior to use (4 volumes of ≥ 95% ethanol per volume of Monarch DNA Wash Buffer)

For 50-prep kit add 20 mL of ethanol to the Monarch DNA Wash Buffer.
For 250-prep kit add 100 mL of ethanol to the Monarch DNA Wash Buffer.

Always keep all buffer bottles tightly closed when not in use.

 




[GUIDELINES]
DNA Cleanup and Concentration: for the purification of up to 5 µg of DNA (ssDNA > 200 nt and dsDNA > 50 bp) from PCR and other enzymatic reactions. A detailed protocol and a quick protocol are available for your convenience. 

Oligonucleotide Cleanup (steps): for the purification of up to 5 µg of DNA fragments ≥ 15 bp (dsDNA) or ≥ 18 nt (ssDNA). Expected recovery is > 70%. When purifying ssDNA of any size, recovery can be increased by using this protocol; however, it is important to note that this protocol shifts the cutoff for smaller fragments to 18 nt (rather than 50 nt for the DNA Cleanup and Concentration Protocol).
A step-by-step protocol is available here.

The Monarch PCR & DNA Cleanup Kit protocol can be modified to enable the purification of ssDNA, oligonucleotides, and other small DNA fragments. The following modified protocol utilizes the same columns and bind/wash/elute workflow of the Monarch PCR & DNA Cleanup Kit with > 70% recovery and cleanup of oligonucleotides ≥ 15 bp (dsDNA) or ≥18 nt (ssDNA). The Oligonucleotide Cleanup protocol efficiently removes unincorporated nucleotides, short oligos, dyes, enzymes, and salts from labeling and other enzymatic reactions.

  
General Guidelines:

Input amount of DNA to be purified should not exceed the binding capacity of the column (5 μg). A starting sample volume of 50 μl is recommended. For smaller samples, nuclease-free water can be used to adjust the volume to the recommended volume range. Centrifugation should be carried out at 16000 x g in a standard laboratory microcentrifuge at room temperature.

[STEPS]
1. Add 100 µL to the 50 µL.
 
2. Add 300 µL. Mix well by pipetting up and down or flicking the tube. Do not vortex.
3. Insert column into collection tube and load sample onto column and close the cap.
4. Spin at 16000 x g for 1 min, then discard flow-through.
 

5. Re-insert column into collection tube.
6. Add 500 µL and spin at 16000 x g for 1 min.
7. Discard flow-through.
 

8. Repeat steps 5-7 (Optional).
 

   
9. Transfer column to a clean 1.5 ml microfuge tube. 
 

10. Add ≥ 6 µL to the center of the matrix.
11. Wait for 1 min, then spin at 16000 x g for 1 min to elute DNA.
TITLE: El-cheep-o bacterial DNA isolation / miniprep
AUTHORS: Magdalena Julkowska

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol for cheep miniprep isolation. Be aware that the DNA quality might not be sufficient to use this protocol for cloning purposes. It works very well for the digestion analysis though. </div></div>

[STEPS]
?. Spin 3ml of the overnight culture to pellet cells (1.5 ml per time) @13000 rpm for 5 min
?. ·Remove the supernatant
?. Add 250 ul of P1 solution + Rnase 5ul and vortex to resuspend the cells
?. Incubate 15 min @ room temperature
?. Add 250 ul of P2 solution and mix gently
?. Incubate 5 min @ room temperature
?. Add 350 ul of P3 solution and mix gently
?. Leave for 10 min on ice
?. Spin @ the max speed for 10 min
?. Take 750 ul of the supernatant and put in a new eppendorf tube. Add 750 ul of isopropanol
?. Mix well
?. Spin for 30 min @ 14000 rpm
?. Remove the supernatant. Wash with 80% EtOH
?. Spin again for other 10 min
?. Dry the pellet @ room temperature for 10 min. Remove the remaining EtOH
?. Add 50 ul of sterile / MiliQ water
?. Measure the DNA concentration using NanoDrop and store the DNA @ -20°C
TITLE: Emma&#39;s Snickerdoodles
AUTHORS: Emma Slayton

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Emma's Snickerdoodles</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">1 cup packed brown sugar</li><li style = "counter-reset:ol0;list-style-type:disc;">2/3 cup white sugar</li><li style = "counter-reset:ol0;list-style-type:disc;">1 cup shortening or butter</li><li style = "counter-reset:ol0;list-style-type:disc;">2 eggs</li><li style = "counter-reset:ol0;list-style-type:disc;">1 1/2 teaspoons vanilla extract</li><li style = "counter-reset:ol0;list-style-type:disc;">2 1/2 cups all-purpose flour</li><li style = "counter-reset:ol0;list-style-type:disc;">1 teaspoon baking soda</li><li style = "counter-reset:ol0;list-style-type:disc;">1 teaspoon salt</li><li style = "counter-reset:ol0;list-style-type:disc;">As much cinimon as you can handle (5 table spoons, or more)</li></ul></div><div class = "text-block">Pre-heat Oven at 350f</div><div class = "text-block">Mix together sugars (1 cup packed brown sugar and 1/2 cup white sugar), shortining or butter, eggs, vanilla untill througly combined. Slowly add in flour, bakingsoda, salt, and cinimon to taste (2 table spoons). </div><div class = "text-block">Once dough is mixed, roll cookies into 1 to 2 inch balls. Roll these balls in a mixture of remaining sugar and cinimon. Place on baking tray, carful to keep cookies far enough apart. </div><div class = "text-block">Bake for 9 to 11 min, or until fully golden in color.  </div></div>

[STEPS]
TITLE: Addgene paper recovery
AUTHORS: Guilherme Barbosa

[DESCRIPTION]
Simple steps to recover plasmid from paper.

[BEFORE_START]
Have the plasmid on paper

[GUIDELINES]
Follow the protocol for simple plasmid recovery from paper

[STEPS]
SECTION: To recover your plasmid on paper:
1. 

Use a clean razor blade to cut out one of the circles containing your DNA. 
 


 

SECTION: To recover your plasmid on paper:
2. Immerse the circle in 30 µL of TE and pipette to mix.

SECTION: To recover your plasmid on paper:
3. After waiting for at least 10 min, use 2 µL to transform competent bacteria.
TITLE: Expression and purification protocol of FIP200 full length or FIP200∆641–779
AUTHORS: Xiaoshan Shi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes the expression and purification of FIP200 full length or FIP200</span><span style = "vertical-align:super;">∆641–779</span><span>.</span></div></div>

[STEPS]
?. [1-    Protein expression:]
Transfect DNA  in cells at a concentration of 2.5–3×106/mL using polyethylenimine (Polysciences) and harvest after  expression, harvest and lyse cells with lysis buffer.
?. [2-    Protein Purification:]
Glutathione Sepharose 4B (GE Healthcare) followed by MBP-tag affinity purifications.Incubate supernatant (added 6M NaCl to a final concentration of 0.5 M) with Glutathione Sepharose 4B resin at  with gentle shaking for , apply to a gravity flow column, and wash extensively with Wash Buffer 1.
4 °C
?. [1-    Protein expression:]
Pellet the harvested cells at  for  at , wash with PBS once, and then store at .
Centrifuge: 500 34
4 °C
-80 °C
?. [1-    Protein expression:]
Resuspend the cell pellet in Lysis Buffer, gentlely shake at  for  and clear it at  at  for .
4 °C
Centrifuge: 16.000 33
4 °C
?. [2-    Protein Purification:]
Elute protein of interest with Elution Buffer 1 and then further apply onto Amylose resin for a second step of affinity purification. Wash Amylose resin with Wash Buffer 2, and elute Protein of interest with Elution Buffer 2.
?. [2-    Protein Purification:]
Pool peak fractions containing pure protein, concentrate, snap-freeze in liquid nitrogen and store at .Yield: about 0.2 mg per litter culture
-80 °C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>sgRNA template assembly, in vitro T7 transcription, and SPRI bead cleanup</p>

[GUIDELINES]
The primers used are: one long, variable oligo that carries the T7 promoter and desired guide sequence; an 82-nt constant oligo that carries the 3' end of the sgRNA; two short external primers for amplification.<br /><strong><br />Assembly Oligos:</strong><br />T7FwdVar: <br />GGATCCTAATACGACTCACTATAG---guide-sequence---GTTTTAGAGCTAGAA <br />T7RevLong: AAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC <br /><br /><strong>Amplification Primers:</strong><br />T7FwdAmp: GGATCCTAATACGACTCACTATAG <br />T7RevAmp: AAAAAAGCACCGACTCGG <br /> <img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/6zc75n.png" alt="" data-src="https://s3.amazonaws.com/pr-journal/6yc75n.png" data-ofn="T7E1 gel.png" /><br /><strong>Designing the guide sequence:</strong><span style="font-weight: 400;">The T7 transcript starts with the G before the dashes in the Fwd assembly oligo. If your target calls for a G at the 5’ end of the guide, use the one already included in T7FwdVar design. If your target calls for a different base at the 5’ end, put the whole guide sequence in T7FwdVar; there will be an extra G on the end that should have minimal effect, unless your guide is quite short.<br /></span><strong>Making the template: </strong>we have used <span style="font-weight: 400;">the Phusion-HF DNA polymerase. Although the amplification primers have quite different Tm’s, they work together fine. </span><br /><br /><br />

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: DNA extraction for HMW DNA
AUTHORS: Natalie Solonenko, Marie Burris

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is intended for extraction of HMW DNA from bacterial or viral samples.</div></div>

[STEPS]
?. [Lyse cells/viruses]
Add 600ul lysis buffer to less than 500ul of sample in a 2mL tube.
[lysis buffer]
To make lysis buffer, add the following to 10mL of 1X TE:1. 300ul 20% SDS2. 60ul 20mg/mL protK
?. [Extract DNA]
Add 1 vol phenol:chloroform and mix by inversion.
Always work with phenol:chloroform in a fume hood.
?. [Extract DNA]
Repeat step 2.
?. [Extract DNA]
Add 1 vol chloroform and mix by inversion.
Always work with chloroform in a fume hood.
?. [Precipitate DNA]
Add ice cold absolute ethanol to fill tube (2.5-3 vol) and mix by inversion.
?. [Precipitate DNA]
Rinse pellet with 1mL RT 70% ethanol.
[70% ethanol]
?. [Lyse cells/viruses]
Incubate 1hr at 37 C.
37 °C
?. [Extract DNA]
Spin at max speed 5 min at RT.
0 Room temperature
?. [Extract DNA]
Remove aqueous layer to new 2mL tube.
?. [Extract DNA]
Spin at max speed 5 min at RT.
0 Room temperature
?. [Extract DNA]
Remove aqueous layer to new 2mL tube.
?. [Precipitate DNA]
Incubate at -20C for >30 min.
-20 °C
Shorter incubation time can be used, but is not recommended for low biomass samples. The longer the incubation the more DNA will precipitate, but note that more salts will precipitate as well.
?. [Precipitate DNA]
Spin at max speed for 15 min at 4 C. Remove and discard supernatant.
4 °C
?. [Precipitate DNA]
Spin at max speed for 2 min.
0 Room temperature
?. [Precipitate DNA]
Remove and discard supernatant.
?. [Precipitate DNA]
Air dry DNA pellet.
This can be done at room temperature, but will go faster at 40 C. Avoid over-drying the pellet as this will make it more difficult to resuspend.
?. [Resuspend DNA]
Resuspend DNA in 100ul warm nuclease-free water.
[water]
Do NOT vortex or pipet to resuspend. Gently flick the tube and leave at room temperature for up to 4 hours. Store at 4 C overnight and flick again the next day. If your pellet is difficult to resuspend, incubate at 56 C flicking periodically until the pellet dissolves.
?. [Resuspend DNA]
At this point you can check the concentration (we recommend Qubit) and purity (NanoDrop) of  your DNA. Ensure the pellet is fully resuspended before you try.