eamag/protocols-with-steps huggingface dataset:
TITLE: Vesicle Fusion on SiO2 Substrates
AUTHORS: Paul Stevenson, Nicole Voce

[DESCRIPTION]
This protocol outlines the steps to produce large area, uniform supported lipid bilayers on SiO2 substrates with or without patterned features.

[STEPS]
SECTION: Substrate cleaning
1. Clean plain SiO2 or TiO2-patterned substrates by sonicating them in a 1:1 IPA:Acetone mixture for ~2 minutes
Dry with a steady stream of N2
SECTION: Substrate cleaning
2. Oxygen plasma clean the substrates for 2 minutes to remove any residual surface contamination
 

SECTION: Vesicle prep
3. Prepare a 1.2 mg/mL DLPC 0.5mol% Rho-PE solution (suitable for FRAP measurements and general fluorescence imaging) or a 0.3mg/mL DLPC 0.0025mol% Rho-PE solution (suitable for FCS measurements)
SECTION: Vesicle prep
3.1. To prepare 1.2 (0.3) mg/mL DLPC 0.5 (0.0025) mol% Rho-PE solutions:

Measure out appropriate amount of DLPC (stored as powder) into a glass vial
 

Add appropriate amount of Rho-PE (stored as 1 mg/mL in chloroform) to DLPC
 

Add 100-200 μL chloroform to vial
  
Dry with N2 so the solution doesn't look glassy-- it should have a dull appearance 

  
Leave in vacuum desiccator overnight to fully remove residual chloroform.




SECTION: Vesicle prep
3.2. After drying, re-hydrate solution with phosphate buffered saline to achieve desired concentrations of 1.2 mg/mL or 0.3 mg/mL 

 

    

SECTION: Vesicle prep
4. Extrude 1.2 (0.3) mg/mL DLPC 0.5 (0.0025) mol% Rho-PE solution 21 times
 
        

SECTION: Vesicle prep
5. After extrusion, aliquot the solution into Eppendorf tubes and store at -20ºC until ready to use
SECTION: Bilayer formation
6. 
 

Set hotplate to 30ºC
Use double-sided kapton tape to adhere a substrate to the bottom of an imaging chamber

  

SECTION: Bilayer formation
7. Place the substrate on the hotplate and deposit 30 μL of 1.2 (0.3) mg/mL DLPC 0.5 (0.0025) mol% Rho-PE solution and 90 μL phosphate buffered saline onto the substrate
Cover the substrate with a pipette box top or something else to protect from dust
Let the substrate incubate for 10 minutes

  

SECTION: Bilayer formation
8. After 10 minutes of incubation, deposit 700 μL of phosphate buffered saline onto the substrate, flooding the imaging chamber
  

SECTION: Bilayer formation
9. Wash the substrate by pipetting 500 μL off and then depositing it back on, moving around the substrate
Repeat 15 times
 

SECTION: Bilayer formation
10. After 15 washing steps, take 500 μL off and depose of it

SECTION: Bilayer formation
11. Roll a coverslip over the imaging chamber
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This lysis buffer is strong enough for lysis most cell lines and wild enough to keep protein interactions. It has been tested on mouse embryonic stem cells, cancer cell lines and overexpressed cells.&nbsp;<br /><br />

[GUIDELINES]
<table style="height: 274px;" width="644">
<tbody>
<tr>
<td>Ingridients</td>
<td>Final Conc.</td>
<td>Volume</td>
<td>Stock Conc.</td>
</tr>
<tr>
<td>TrisHCl (PH8.0)</td>
<td>20 mM</td>
<td>100 ml</td>
<td>100 mM</td>
</tr>
<tr>
<td>NaCl</td>
<td>300 mM</td>
<td>30 ml</td>
<td>5 M</td>
</tr>
<tr>
<td>EDTA</td>
<td>0.5 mM</td>
<td>0.5 ml</td>
<td>500 mM</td>
</tr>
<tr>
<td>Na3VO4</td>
<td>1 mM</td>
<td>5 ml</td>
<td>100 mM</td>
</tr>
<tr>
<td>NaF</td>
<td>50 mM</td>
<td>50 ml</td>
<td>500 mM</td>
</tr>
<tr>
<td>NP-40</td>
<td>1 %</td>
<td>5 ml</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Triton-X100</td>
<td>1 %</td>
<td>5 ml</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>H2O</td>
<td>&nbsp;</td>
<td>304.5 ml</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>Total</td>
<td>&nbsp;</td>
<td>500 ml</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>
<br />After mix, sterilize it by filter through 0.2&nbsp;&mu;m filter. Store at 4&nbsp;&deg;C. Add protease inhibitor cocktails just before using.

[STEPS]
TITLE: HMW DNA extraction for amphipods
AUTHORS: Benoît   Vacherie, Karine  Labadie

[DESCRIPTION]
Protocol adapted from Qiagen's genomic DNA handbook protocol for the HMW extraction of live or flash-freezed amphipods.

[GUIDELINES]
To preserve large DNA sizes, never use Vortex and only use Wide-Bore tips.

[STEPS]
SECTION: Tissue homogenization
1. Prepare the lysis buffer by adding  3 µL of RNase A (100 mg/mL) to 1.5 ml of Buffer G2 per sample.
  
  

SECTION: Tissue homogenization
2. place live or flash-freezed amphipod in 2ml douncer and add 1 ml of lysis buffer
SECTION: Tissue homogenization
3. gently up and down 10 times with the piston
SECTION: Tissue homogenization
4. transfer the lysate to a 2 ml tube
  

SECTION: Tissue homogenization
5. rinse the douncer with 0.5 ml lysis buffer and transfer to the 2 ml tube
SECTION: Lysis
6. Incubate for 30 minutes at 37°C.
37 °C 30 min 

SECTION: Lysis
7. Add 75 µL of Proteinase K (20 mg/mL) and incubate with inversion at 50°C for 2 hours.
  
120 min 50 °C 

SECTION: Lysis
8. Gently transfer in a new 2 ml tube the supernatant with a 1000 µl wide-bore tip, avoiding touching the pelleted debris by gravity.
  

SECTION: DNA binding and washing
9. Place a genomic-tip 20G column on a 15 ml tube
  
  

SECTION: DNA binding and washing
10. Equilibrate the column with 1 mL Buffer QBT. Wait for the Genomic-tip to drain by gravity.
  

SECTION: DNA binding and washing
11. Carefully apply the lysate to the Genomic-tip with a 1000 µl wide-bore tip. Wait for the Genomic-tip to drain by gravity.

SECTION: DNA binding and washing
12. Wash the QIAGEN Genomic-tip with  3 x 1 mL Buffer QC.
  

SECTION: DNA binding and washing
13. Elute the DNA into a new 15 mL tube with 2 x 1  mL of Buffer QF prewarm to 50°C
  

SECTION: DNA recovery
14. Add 1.4 mL of room-temperature isopropanol to the éluate, mix by inversion about 10 times, and centrifuge for 30 min at 5,500g at 4°C to pellet DNA. set centrifuge deceleration speed to level3
  
5500 x g, 30 min, 4 °C 

SECTION: DNA recovery
15. Gently remove the supernatant using a P1000
Gently resuspend the pellet with 1 ml of cold 70% ethanol using a 1000µl wide-bore tip.
Transfer to a new 1.5 ml tube.
  
  

SECTION: DNA recovery
16. Centrifuge for 10 min at 5,000g at 4°C to pellet DNA. set centrifuge deceleration speed to level3
5000 x g, 10 min, 4 °C 

SECTION: DNA recovery
17. Remove as much supernatant as possible, avoiding touching the pellet.
Air dry the pellet 10 min at RT.
10 minRoom temperature 

SECTION: DNA recovery
18. Resuspend the pellet with 50 µL of TE 1x prewarm to 50°C
  

SECTION: DNA recovery
19. Incubate for 1hour at 50°C, then overnight at RT.
60 min 50 °C 
  Room temperature 

SECTION: Sample QC
20. Quantify your sample with a Qubit HS. 

Analyse 1 µL in a UV spectrophotometer (e.g. Nanodrop).

Visualise 1 µL of sample to estimate the molecular weight.  (Tapestation)


SECTION: Results
21. QC results of extraction of 4 individuals
 

SECTION: Results
22. Tapestation profiles
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is adapted from the Invitrogen Life Technologies Trizol manual. </p>

[BEFORE_START]
<ol>
<li>Prepare an <strong>RNase-free</strong> working area, wipe down barrels of micropipettes, use <strong>filter tips</strong> and RNase-free microcentrifuge tubes, and always wear <strong>gloves</strong>. </li>
<li><strong>Snap-freeze tissue</strong> as it is collected by adding tissue to a microfuge tube on dry ice.</li>
<li>This protocol calls for <strong>50-100 mg of tissue</strong> to be homogenized in a microcentrifuge tube in <strong>1 mL of TRIzol Reagent</strong>. *Use caution here - homogenization with a motorized homogenizer may result in overflow. </li>
</ol>

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

[DESCRIPTION]
<p><em>The MELD Project is an international collaboration aiming to create open-access, robust and generalisable tools for FCD detection. To this end, we will train a neural network classifier on MRI features from FCD patients from multiple centres worldwide.</em></p>
<p><strong> </strong></p>
<p><strong>Protocol 1 details instructions for patient and control inclusion in the MELD Project as well as how to fill out the demographics csv file. </strong></p>

[BEFORE_START]
<p>Ensure that you have "cloned" the MELD file structure from github. (https://github.com/MELDProject/meld)</p>
<p> </p>
<p>To do this, in a terminal window cd into the location you wish to store the data.</p>
<p>`cd &lt;path&gt;`</p>
<p>Then "clone" the repository using the following command:</p>
<p> </p>
<p>git clone https://github.com/MELDProject/meld</p>
<p> </p>
<p>This will download all of the scripts, template folder structure and template control data necessary for the MELD preprocessing.</p>
<p> </p>
<p>This contains the MELD_site_code_participants.csv file that is required for this protocol.</p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>

[GUIDELINES]
<p><strong>If you have any questions or run into problems, please feel free to contact the MELD project: (meld.study@gmail.com)</strong></p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Lightsheet Tissue Intake - Photodocumentation and Tracking
AUTHORS: Seth Currlin, Marda Jorgensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>In order to track tissue position and orientation each sample is photographed when received for lightsheet microscopy (Figure 1, A). Large samples (>1 cm</span><span style = "vertical-align:super;">3</span><span>), such as the spleen in Figure 1, are cut into 2 mm sections (Figure 1, B) to facilitate tissue clearing, staining, and three-dimensional imaging. These tissues are batch processed and withdrawn from the lipid clearing pipeline as needed (Figure 1, C); at this time a novel identifier is assigned to the sample. </span></div></div>

[STEPS]
?. Photograph whole tissue; capture from various angles if possible. A dissection ruler or grid should accompany each photograph.
?. Using a razor blade and forceps slice the tissue into sections approximately 2 mm wide. Try to slice in a continuous motion, while applying a gentle downward force.
?. Set aside tissue section and repeat Step 2 as needed. Maintain or keep track of slice order and orientation.
?. Photograph the sequential 2 mm slices, again using a dissection ruler or grid.
?. Retain all slices within one container for subsequent processing (lipid removal).
TITLE: Low Biomass, high contamination Illumina DNA prep using DNeasy PowerSoil (Pro) Kit
AUTHORS: Tania Kurbessoian, Jason E Stajich, Sonia L. Ghose

[DESCRIPTION]
This is an addendum to the already optimized DNeasy PowerSoil Kit. This can also be applied to the PowerSoil Pro Kit.
Use this protocol for Low Biomass or high contamination samples.



[BEFORE_START]
Make sure you have all the components of the kit ready to go. 

[GUIDELINES]
Using some small tweaks to an already optimized protocol you can get your DNA from contaminated samples!

[STEPS]
1. Add sample into the PowerBead tube.
2. Add 60μL of Solution C1. 
2.1. Make sure the sample has not precipitated. If so, heat sample to 60ºC until precipitate is dissolved into solution. 

3. Vortex to mix and incubate at 65ºC for 10 minutes.
4. Bead beat at "homogenize" setting: 90 seconds bead beating, 60 seconds rest, then another 90 seconds (3 minutes total of actual beating).
5. Centrifuge at 10,000 x g for 30 s.
6. Remove the supernatant or upper aqueous layer if you have one, to the new tube.
7. Add 100 uL Solution C2 and 100μL of Solution C3. Vortex to mix. Incubate at 4ºC or on ice for 5 minutes.
7.1. *This is for low humic soils (100μL of each). If high humic soil, add 150μL C2 followed by 150μL C3.
8. Centrifuge to pellet (1 min at 10,000 x g.) and transfer the supernatant to a new tube. Avoid pellet.
9. Ideally you will have 650μL of lysate, and can add 650μL of Solution C4 and 650μL of 100% ethanol.
9.2. If you have 700μL, add 700μL of Solution C4 and 600μL of 100% ethanol. (Keep it as close to 1:1 as possible)
10. Load the lysate to the spin filter 650μL at a time and bind in three steps, alternating with centrifugation (1 min at 10,000 x g). Discard flow-through each time.
9.1. Make sure you mix C4 solution well. If new kit add ethanol.
11. If the membrane is not stained brown, wash with 650μL of 100% ethanol (centrifuge 1 min at 10,000 x g and discard flow-through).
11.1. If the membrane is stained brown, per sample, prepare a mix of 300μL of Solution C4 and 370μL of 100%
ethanol. Wash the column with this mixture first. Follow this wash with the 100% ethanol wash.
12. Wash with 500μL of solution C5 (centrifuge 1 min at 10,000 x g and discard flow-through)
13. Dry the spin column for 2 minutes 10,000 x g. Transfer to a clean tube.
14. Elute in 60μL of buffer C6 (Heat this buffer to 60ºC before eluting). Let the buffer sit on the membrane 5
minutes before elution. 
15. Then centrifuge for 1 min 10,000 x g into your storage tube.
TITLE: Aggregation propensity of α-synuclein seeds in primary neurons
AUTHORS: Arpine Sokratian

[DESCRIPTION]
This protocol describes the details of the treatment of primary hippocampal culture with α-synuclein fibrils or monomer protein. It also includes details of the collection of cell lysates for immunoblotting or ELISA measurements. Another part of the protocol describes immunostaining analysis of treated neurons to measure the seeding propensity of α-synuclein fibrils visualized with phospho-S129 α-synuclein antibodies.

[STEPS]
1. Prepare primary hippocampal neurons according to protocol 
 

3.  At DIV21 for 14 days of incubation or at DIV14 for 7 days of incubation culture media should be removed - aspirate the media
4. Here are options to use the prepared cultures for the western-blot analysis or ELISA (step-case) or for the immunofluorescence
5. Spin down a tube of normal donkey serum (20k xg for 10 min, 4C). Remove supernatant, do not use pellet.
6. Rinse with 3x with TBS.  Use plastic bulb dropper and be careful. Prefer to remove w/ 200uL pipette, not vaccum. DO NOT let cells dry out, only do a row at a time.
7. Block/ Permeabilize with 300 uL 0.1% saponin, 3% Normal Donkey serum in TBS 30 min RT [KEEP COVERED]
8. During block, prepare primary antibodies:
Add primary antibody at desired concentration with a supplement of 0.02% saponin, 1% donkey serum, in TBS. Spin down the tube (20k xg for 10 min, 4C). Remove supernatant, do not use pellet

 MJFR14 (EPY, 1:4,000), Tau5 (1:2,000), NeuN (1:2,000) 
9. Remove block from wells.
Add primary antibody solutions to well. (300uL)
Overnight at 4C. Gentle rocking (no orbital shaker). Cover in Foil and make sure no loss of liquid. 
10. Rinse with 1x with 300uL of TBS
Prepare secondary antibodies. 
Use anti-rabbit AlexaFlour 647, anti-mouse AlexaFlour 488, anti-chicken AlexaFlour 555 at 1:1000 concentration, supplemented with 0.02% saponin, 0.1% normal  goat serum in PBS. Spin down the tube (20k xg for 10 min, 4C). Remove supernatant, do not use pellet. Add Hoescht 1:10,000 to staining buffer
11. Rinse two times with TBS
Add TBS imaging buffer, take pics on Keyence.
Imaging: let the plate come up to RT, wipe bottom of plate with 70% ethanol before imaging
Take x25 20x pictures per well.  
 Images were obtained using Keyence BZ-X810 and Zeiss880 and coded for a blinded approach to analyze using CellProfiler and imageJ. 
2. at DIV7 treat the culture with α-synuclein fibrils/seeds or monomeric protein at concentration 0.64 nM or 64 pM relative to estimated molecular weight measured as calculated size by DLS acquisitions and protein weight denatured in 6M GuHCL and calculated via Nanodrop.
TITLE: GM Covid-19 saliva test (v2)
AUTHORS:  

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">GM Covid-19 saliva test is a RT-PCR test intended for qualitative detection of nucleic acids from SARS-CoV-2 in saliva specimen. The test is used for screening purpose under class of "research used only" (RUO). The test is not for diagnostic purpose. Positive result would be referred to an official clinical test with appropriate certification (i.e CLIA) required for clinical diagnosis.</div></div>

[STEPS]
?. [Setup reaction]
Prepare PCR master mixPrepare 100 reactions by adding 1ml Buffer into 1ml Enzyme. Aliquot 20ul into each PCR tube.
?. [Setup reaction]
Saliva-PrepSaliva is self-collected into any sterile tubes with screw cap and wide opening (Sanitize hands before and after saliva collection); Use disposable transfer pipette with fixed volume to pipett  saliva into a Saliva-Prep tube, tightly screw cap, and mix thoroughly;Heat the Saliva-Prep tube at  for   by using a water bath or dry-bath;Keep the tube in refrigerator for  , then on GM-rack for
70 °C
?. [RT-PCR]
Load sampleUse disposable GM-stick to slowly insert into Saliva-prep tube and slowly pull it out, then directly dip it into a PCR tube. Close PCR tube. Repeat this step for each sample.Pulse spin the PCR strip/plate to bring the liquid to bottom.
?. [RT-PCR]
RT-PCRPut the PCR strip/plate into a thermal cycler, choose and start the program "GM Covid19 Test".Note: The test is suitable for conventioinal thermal cylers. BenchMark's TC32 and Thermofisher's Veriti thermocycler have been tested.
?. Check color Check color of PCR reactions while keeping PCR tube closed. There are two ways to check color of PCR reactions:
TITLE: Structured Interview for Protocol Use
AUTHORS: Drew Gitomer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a modification of Palinkas et al SIEU.  Based on a lot of feelings and intuitions we thought that a 4 point scale would be super.</div><div class = "text-block">This article describes the Standard Interview for Evidence Use (SIEU), a measure to assess the level of engagement in acquiring, evaluating, and applying research evidence in health and social service settings. Method: Three scales measuring input, process, and output of research evidence and eight subscales were identified using principal axis factor analysis and parallel analysis of data collected from 202 state and county child welfare, mental health, and juvenile justice systems leaders. Results: The SIEU scales and subscales demonstrate strong internal consistency as well as convergent and discriminant validity. Conclusions: The SIEU is easy to use and can be administered as a complete scale or as three smaller scales to separately examine evidence in acquisition, eva-luation, or application. The measure demonstrates potential in understanding the role of research evidence in service settings and in monitoring the process of evidence-based practice and application of scientific principles in social work practice.</div></div>

[STEPS]
TITLE: ssDNA2.0: Fill-in mix
AUTHORS: Sarah Nagel, Anna Schmidt, Matthias Meyer

[DESCRIPTION]
Protocol for the preparation of Fill-in mix for automated single-stranded DNA library preparation using the ssDNA2.0 method (Gansauge et al. 2020). 

References
Gansauge, M.-T., Aximu-Petri, A., Nagel, S., & Meyer, M. (2020). Manual and automated preparation of single-stranded DNA libraries for the sequencing of DNA from ancient biological remains and other sources of highly degraded DNA. Nature Protocols, 15, 2279-2300.

[STEPS]
TITLE: Procedure for measuring extraction efficiency
AUTHORS: Krista Longnecker, Gretchen Swarr

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Capturing dissolved organic matter from seawater using solid phase extraction is not 100% efficient. We use this protocol to quantify the amount of organic carbon retained during the extraction step. Comparing this value to the concentration of dissolved organic carbon in the initial water sample allows us to quantify the efficiency of the extraction procedure.</div></div>

[STEPS]
?. [Procedure for measuring extraction efficiency]
This protocol starts with the eluent from a solid phase extraction resin. Dry down eluents in vials (all the way down, no solvent remaining!).
8 mL
?. [Procedure for measuring extraction efficiency]
While drying down prepare  EPA vials with  MQ water and  HCl.
40 mL
25 mL
25 µl
12
?. [Procedure for measuring extraction efficiency]
Once the  vials have dried down fully add  of MQ water and vortex.
8 mL
1 mL
?. [Procedure for measuring extraction efficiency]
Add  of this vortexed solution to your prepped  EPA vials. The final volume should be .
400 µl
40 mL
25.425 mL
?. [Procedure for measuring extraction efficiency]
Load the  vial on to the autosampler for the TOC analyzer ( is injected onto the column).
40 mL
150 µl
?. [Procedure for measuring extraction efficiency]
Once these are done, dry down the remaining solution in the  vials for long term storage.
8 mL
Extraction efficiency calculation:Note that this calculation assumes that you have dried down the entire extract for this analysis and that no sample has been removed from the extract for other analyses.Example for 466 μM NPOC in DOC (not TOC) sample and 240 ml total volume of extract:(240 ml) (466 μM) = (1 ml) (x)x = 111,840 μM in extract if 100% efficientActual measured carbon in extract example (for sample with 46 μM measured carbon):(400 μl) (x) = (46 μM) (25 ml) (1000 μl / 1 ml)x = 2875 μM% efficiency = 2875 μM / 111,840 μM =
TITLE: Chloroform-free  DNA Extraction - Ammonium Acetate Precipitation Method
AUTHORS: NERC Environmental Omics Facility (NEOF) Visitor Facility 

[DESCRIPTION]
A chloroform-free, cost-effective DNA extraction method for a variety of sample types.

[STEPS]
1. Add 250 µL Digsol buffer (see Materials for recipe) and 10 µL   to a lablelled 1.5ml tube.

2. Tissue: Cut into small pieces (<1cm2) with a sterile razor blade on a sterile glass plate before adding to the 1.5mL tube.
Blood: Centrifuge blood sample at 13,000rpm for about 1 min (to pellet sample).
Remove sample from ethanol with toothpick and blot onto tissue. When almost dry, transfer the toothpick into the 1.5mL tube and jiggle to dislodge the blood. Remove toothpick and place in disinfectant.
Swab: Dry of ethanol and place into the 1.5mL tube. Snap off the end of the swap so the lid may close.
Feather: Cut the calamus of 1-3 feathers into small pieces with a sterile razor blade on a sterile glass plate before adding to the tube. If feathers are very small and blood spots are present on the tips, feathers can be added whole.
Hair: Place hair(s) into the 1.5mL tube preferably with the largest root and add 5 µL   
Insect: Degut and add to 1.5mL tube. If the insect is very small, starve for 48hrs before freezing. Add 5 µL  . Crush with a pestle or add a metal lysing bead and place on TissueLyser (Qiagen) for 2min, 30/s. 
 

3. Vortex and place samples in a rotating oven at 55 °C for 180 min or   for maximum digestion.

4. Add 300 µL 4M .

5. Vortex several times over a period of at least 15 min at Room temperature.

6. 13000 rpm, 10 min 

7. Aspirate the supernatant (clear liquid containing the DNA) into a new labelled 1.5ml tube. Discard tube containing the pelleted protein debris.
8. Add 1 mL 100% ethanol and invert the tube gently several times to precipitate DNA.

9. 13000 rpm, 10 min 

10. Pour off ethanol in a smooth motion, taking care not to lose the DNA pellet.
11. Add 500 µL 70% ethanol and invert gently several times to clean the pellet.

12. 15000 rpm, 5 min 

13. Pour off ethanol in a smooth movement or using a pipette gently draw off the supernatant if fear of losing the pellet. Stand tubes upside-down on clean tissue until dry (approx. 30-60 minutes). This can be sped up by using the heat of a lamp from above.
14. Once fully dry add approx. 100 µL Low TE (see Materials for recipe). Add less if a very tiny pellet or no pellet is observed. Flick sample to dislodge pellet.

15. Place tubes in a thermomixer or shaking oven at 50 °C  for 30 min to dissolve the pellet. If the pellet has not completely dissolved add more Low TE.

16. Store at -20 °C (long term) or 4 °C (short term).