eamag/protocols-with-steps huggingface dataset:
TITLE: HMW gDNA extraction from prokaryotic cultures and cryo preservation stocks
AUTHORS: Richard RKS Stöckl

[DESCRIPTION]
This protocol can be used to extract High Molecular Weight gDNA from bacterial and archaeal cultures and cryo preservations thereof. 
The resulting gDNA is usually suitable for long-read sequencing and is regularly used for genome assembly following Nanopore Sequencing.
It has been tested with a variety of mostly archaeal but also bacterial strains, including, but not limited to, Thermococcales, Thermotogales, E. coli, and Desulfurococcales.

[STEPS]
SECTION: Prepare cultures or cryopreservation capillaries
1. Transfer the bacteria-/archaea-suspension (~50 µL) from one cryo preservation capillary to a 1.5 mL reaction tube or pellet a well-grown culture by centrifugation 15000 rpm, 15 min , discarding the supernatant, and resuspending the cell pellet in ~50 µL media

SECTION: Open up the cells
2. Add 490 µL   + 20 µL freshly prepared    solution (10 mg/mL, in  ) and vortex briefly

SECTION: Open up the cells
3. incubate 30 min at 37 °C

SECTION: Open up the cells
4. add 5 µL   (20 mg/mL) + 10 µL   (10 mg/mL) and 15 µL of 20 Mass / % volume   , vortex briefly

SECTION: Open up the cells
5. incubate 60 min at 56 °C

SECTION: Open up the cells
6. freeze at -80 °C for 30 min and thaw at 60 °C for 10 min

SECTION: Open up the cells
7. repeat   two additional times (total of three cycles)

SECTION: Open up the cells
8. add 100 µL of 5 Molarity (M)    and mix well
 

SECTION: Open up the cells
9. add 80 µL of 10 Mass / % volume   in 700 millimolar (mM)  

SECTION: Open up the cells
10. incubate at 65 °C for 30 min

SECTION: Remove non-DNA components
11. add one volume (should be roughly 750 µL) chloroform:isoamyl alcohol (24:1;   ), shake vigorously, centrifuge full speed (> 12.000 rcf, 2 min), transfer top (aqueous) phase to new tube

SECTION: Remove non-DNA components
12. mix aqueous phase with equal volume phenol:chloroform:isoamyl alcohol (25:24:1;   ), centrifuge full speed (>12.000 rcf, 2 min) as before, and transfer top (aqueous) phase to new tube

SECTION: Remove non-DNA components
13. mix aqueous phase with equal volume chloroform:isoamyl alcohol (24:1;   ), centrifuge as before (>12.000 rcf, 2 min), and transfer top (aqueous) phase to new  

SECTION: Pellet and wash gDNA
14. add 0.6 volumes (roughly 360 µL)  of cold 100%   + 0.06 volumes (roughly 36 µL)  of 3 Molarity (m)   pH 5.2

SECTION: Pellet and wash gDNA
15. incubate at -20 °C overnight

SECTION: Pellet and wash gDNA
16. centrifuge 16.000 rcf, 20 min , discard supernatant, air dry briefly

SECTION: Pellet and wash gDNA
17. add 0.6 volumes  cold (-20 °C) 70 % (v/v)   , centrifuge at 16.000 rcf, 10 min , discard supernatant, air dry briefly

SECTION: Pellet and wash gDNA
19. resuspend in 50 µL   or   or similar (ideally let this rest at 4 °C for several hours)

SECTION: Pellet and wash gDNA
18. Optional: repeat   once
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The Campbell Lab protocol for imaging Western Blots using ECL Select chemi-luminescent detection and the Bio-Rad VersaDoc imager for immunoquantitation of phytoplankton samples.</p>

[GUIDELINES]
<p>The ECL Select is not the most sensitive detection reagent we've tested (that would be ECL Ultra from Lumigen) but it has a linear reaction profile over time when doing quantitative immunoblotting.</p>

[STEPS]
TITLE: DNA yield estimation and custom DNA quality evaluation before shotgun metagenomic sequencing
AUTHORS: Benoit Quinquis , Alexandre Famechon , nathalie.galleron, Victoria Meslier, mathieu.almeida

[DESCRIPTION]
This protocol describes the procedures used for DNA quantification and a custom DNA quality evaluation before proceeding to shotgun metagenomic seqeuencing. 

[BEFORE_START]
NA

[GUIDELINES]
NA

[STEPS]
SECTION: DNA quantification
1. using the DNA-binding fluorescent Quant-it dsDNA
BR assay 
SECTION: Custom DNA quality estimation
2. Genomic 50 kb kit by capillary electrophoresis using the Fragment Analyzer, followed by a custom DNA quality analysis
TITLE: Seed Sterilization
AUTHORS: Alex Rajewski

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details how to surface sterilize plant seeds for tissue culture regeneration/transformation or for other techniques requiring sterile seeds.</div></div>

[STEPS]
?. Place a 100mL beaker containing approximatelyin the chaber next to the petri dish of seeds.
[bleach]
?. In a fume hood, place an empty dessication chamber with a petri dish of the seeds you wish to surface sterilize. Leave the lid of the petri dish halfway on, so that you can quickly close it later.
A dessication chamber per se is not necessary; you simply need some container to hold the gas for a few hours. It does not need to be thoroughly airtight, but it helps.If you plan to sterilize multiple genotypes of seeds, be aware that the gas produced will erase most markers. Pencil and paper labels work well though.
?. Prepare approximately in a transfer pipet or similar.
[HCl]
?. The following steps must occur quickly to prevent the escape of the gas. Hold the lid to the chamber in one hand. With the other hand squirt the HCl into the beaker of bleach and quickly close the chamber to seal in the gas.
The HCl and the bleach will react violently to produce the gas, do not be startled. The larger beaker should prevent any bubbles of bleach or HCl from contacting the rest of the set up.
?. Allow the seeds to be sterilized for approximately 3 hours. This time works for tomato, tobacco, and Arabidopsis seeds. The seeds may become whiter in color, this is also normal and does not seem to affect germination rates.
?. Once the time is up, quickly open the chamber and slide the lid completely onto the petri dish to seal it. The seeds can be stored like this for several weeks or transfered to a laminar flow hood for further use. Allow the chmaber to air out for an hour or so.
?. If the reaction of the bleach and HCl went to perfect completion, the beaker should only contain salt water. In reality it is likely acidic. Dispose of this according to your local regulations.
TITLE: UC Davis - Protein Carbonyl
AUTHORS: Peter Havel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">
Cayman Chemical's Protein Carbonyl Colorimetric Assay Kit is a convenient colorimetric assay for the measurement of oxidized proteins. Protein samples are derivatized by making use of the reaction between 2,4-dinitrophenylhydrazine (DNPH) and protein carbonyls. Formation of a Schiff base produces the corresponding hydrazone which can be analyzed spectrophotometrically at 360-385 nm. This assay can be used to measure oxidized protein in plasma, serum, cell lysates, and tissue homogenates.</div></div>

[STEPS]
?. Transfer 200 µl of sample to two 2 ml plastic tubes. One tube will be the sample tube (S#) and the other will be the control tube (C#).
?. Add 800 µl of DNPH to the sample tube and add 800 µl of 2.5 M HCI to the control tube.
?. Incubate both tubes (S# & C#) in the dark at room temperature for one hour. Vortex each tube briefly every 15 minutes during the incubation.
?. Add 1 ml of 20% TCA to each tube and vortex. Place tubes on ice and incubate for five minutes.
?. Centrifuge tubes at 10,000 xg for 10 minutes at 4ºC in a microcentrifuge.
?. Discard the supernatant and resuspend the pellet in 1 ml of 10% TCA. Place tubes on ice and let sit for five minutes.
?. Centrifuge tubes at 10,000 xg for 10 minutes at 4ºC in a microcentrifuge.
?. Discard the supernatant and resuspend the pellet in 1 ml of (1:1) Ethanol/Ethyl Acetate mixture. Manually suspend pellet with spatula, vortex thoroughly, and centrifuge tubes at 10,000 xg for 10 minutes at 4ºC in a microcentrifuge.
?. Repeat Step 8 two more times.
?. After the final wash, resuspend the protein pellets in 500µl  of guanidine hydrochloride by vortexing.
?. Centriguge tubes at 10,000 xg for 10 minutes at 4ºC in a microcentrifuge to remove any left over debris.
?. Transfer 220 µl of suspernatant from the sample (S#) tube to two wells of the 96-well plate.
?. Transfer 220 µl of supernatant from the control (C#) tube to two wells of the 96-well plate.
?. Measure the absorbance at a wavelength between 360-385 nm using a plate reader.
?. Calculation1. Calculate the average absorbance of each sample and control.2. Subtract the average absorbance of the controls from the average absorbance of the samples. This is the Corrected Absorbance (CA).3. Determine the concentration of the carbonyls by inserting the corrected absorbance into the following equation:*The actual extinction coefficient for dinitrophenylhydrazine at 370 nm is 22,000 M-1cm-1 (0.022 µM-1cm-1). This value has been adjusted for the pathlength of the solution in the well.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol can be used to transfer total RNA from thick formaldehyde/agarose gels to Nylon membranes for downstream Northern Blot Analysis. This is useful for analyzing larger RNA molecules such as mRNA.</p>

[GUIDELINES]
<p>Always wear gloves to prevent degradation of RNA.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Typical Protocol for NEBExpress GamS Nuclease Inhibitor when used with the NEBExpress Cell-free E. coli Protein Synthesis System (NEB #E5360)
AUTHORS: New England Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>NEBExpress GamS Nuclease Inhibitor is a recombinant protein that inhibits Exonuclease V (RecBCD) activity and stabilizes linear DNA templates in </span><span style = "font-style:italic;">E. coli  </span><span>based </span><span style = "font-style:italic;">in vitro  </span><span>protein synthesis reactions.  </span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;"><span>Enhances synthesis yield of linear DNA using the NEBExpress Cell-free</span><span style = "font-style:italic;">E. coli</span><span>Protein Synthesis System</span></li><li style = "counter-reset:ol0;list-style-type:disc;">Recombinant enzyme; ≥ 95% purity, as determined by SDS-PAGE </li><li style = "counter-reset:ol0;list-style-type:disc;">No detectable protease activity, ensuring stability of desired target protein</li><li style = "counter-reset:ol0;list-style-type:disc;">Compatible with various target proteins, ranging from 17 to 230 kDa</li><li style = "counter-reset:ol0;list-style-type:disc;">Optimal storage buffer for performance in protein synthesis reactions</li><li style = "counter-reset:ol0;list-style-type:disc;">Flexible reaction conditions achieve maximum yield; activity can be sustained for 10 hours at 37°C or up to 24 hours at lower temperatures</li></ul></div></div>

[STEPS]
?. Thaw all components .
on ice
?. Gently vortex the NEBExpress S30 Synthesis Extract and Protein Synthesis Buffer to mix.
?. Combine reagents in a 1.5 ml microcentrifuge tube  as follows:  ABCD1COMPONENTSNEGATIVE CONTROL (no DNA)POSITIVE CONTROLSAMPLE2NEBExpress S30 Synthesis Extract12 µl12 µl12 µl3Protein Synthesis Buffer (2X)25 µl25 µl25 µl4T7 RNA Polymerase1 µl1 µl1 µl5RNase Inhibitor, Murine1 µl1 µl1 µl6DHFR-His control template (125 ng/ µl)--2 µl--7Linear DNA template (>100ng/µL)----250 ng8NEBExpress GamS Nuclease Inhibitor----1 μl9Water11 µl9 µlto 50 µl
on ice
ABCD1COMPONENTSNEGATIVE CONTROL (no DNA)POSITIVE CONTROLSAMPLE2NEBExpress S30 Synthesis Extract12 µl12 µl12 µl3Protein Synthesis Buffer (2X)25 µl25 µl25 µl4T7 RNA Polymerase1 µl1 µl1 µl5RNase Inhibitor, Murine1 µl1 µl1 µl6DHFR-His control template (125 ng/ µl)--2 µl--7Linear DNA template (>100ng/µL)----250 ng8NEBExpress GamS Nuclease Inhibitor----1 μl9Water11 µl9 µlto 50 µl
During the experimental setup, it is recommended to add the linear DNA template in the last step to allow GamS to bind and inhibit RecBCD exonuclease before RecBCD has a chance to act on the DNA. Optimal GamS concentration must be determined empirically for a particular target.
?. Incubate reactions at , with shaking, for  – .
37 °C
Additional incubation time (maximum 10 hours) at 37°C may increase yield.
?. Analyze by method of choice or freeze at  for later use.
-20 °C
TITLE: Modified Promega Wizard Extraction for Barcoding Macrofungi
AUTHORS: Stephen       Douglas Russell

[DESCRIPTION]
This protocol is best used when preparing macrofungal specimens for Sanger sequencing or as a secondary extraction protocol for ONT nanopore barcoding. 

[STEPS]
1. Add 600uL of   to 1.5mL eppi tubes. One tube for each specimen you are planning an extraction for.

2. Place tissue from your specimens into each tube using tweezers. Utilize a piece about the size of a grain of rice. The tissue can be either fresh or dried. Label the tube with the appropriate number. Wipe the tweezers off with a Kimwipe or paper towel in between each specimen. These tubes can be stored at room temperature until they are ready to be used. 
3. Grind the tissue in each tube using a sterile pestle.
4. Heat the tubes at 65 °C for 15 min.

5. Centrifuge the tubes for 3 min.

6. Transfer the supernatant (liquid on top) to a new 1.5mL eppi tube. 

Add 200 µL of   to the tube. 

Vortex the tube for 20 s.

Centrifuge the tube for 6 min.


7. Transfer the supernatant (liquid on top) to a new 1.5mL eppi tube. 

Add 600 µL of 100%   to the tube.  This precipitates the DNA.
 
Centrifuge the tube for 1 min. The DNA will now be in a pellet stuck to the bottom of the tube.

Discard the supernatant. It can just be poured out of the tube into a waste container.

8. Add 600 µL of 70% ethanol to the tube.  

Centrifuge the tube for 1 min.

Discard the supernatant. It can just be poured directly out of the tube into a waste container.

Place the tube upside down on a Kimwipe for at least 15 min, or until all of the ethanol has evaporated from the tube. I usually leave the tube to dry overnight.

9. Add 30uL of molecular water to the tube.

Your DNA template is now ready for amplification.
TITLE: TS Spurr&#39;s - primary fixation Karnovsky&#39;s - tissue (TM - 013)
AUTHORS: sandra.crameri

[DESCRIPTION]
This method is used for conventional processing of tissue to Spurr's resin.

[GUIDELINES]
All time are minimum times, it is acceptable to go over time specified for any given step. Good place steps to leave overnight or at 70% ethanol and 50/50 Spurrs/Ethanol mix.

[STEPS]
SECTION: HEADER
1. SAN:


SPEC No:


OPERATOR & STEPS:


OPERATOR & STEPS:
SECTION: CONVENTIONAL
2. 2.5 % volume   plus 4 % volume    in 0.1 Molarity (m) for at least 40 min 

SECTION: CONVENTIONAL
3. Wash 0.1 Molarity (M) Sorenson's Phosphate Buffer pH 07.2 (300mosmol/kg) for 15 min 

SECTION: CONVENTIONAL
4. 1 % volume   in buffer for 60 min 

SECTION: CONVENTIONAL
5. 70 % volume   for at least 20 min 

SECTION: CONVENTIONAL
6. 95 % volume   for 20 min 

SECTION: CONVENTIONAL
7. 100 % volume   for 20 min 

SECTION: CONVENTIONAL
8. 100 % volume   dried molecular sieved for 20 min 

SECTION: CONVENTIONAL
9. 50 % volume   50 % volume   for 30 min 


SECTION: CONVENTIONAL
10. 100 % volume   30 min 

SECTION: CONVENTIONAL
11. 100 % volume   60 min 

SECTION: CONVENTIONAL
12. Oven polymerise overnight at 65 °C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For use in <a href="https://www.protocols.io/view/Wet-mount-Method-for-Enumeration-of-Aquatic-Viruse-c8pzvm" target="_blank">Wet-mount Method for Enumeration of Aquatic Viruses</a>

[GUIDELINES]
<strong>Note:</strong> An alternative Ascorbate-EDTA Buffer can be made with MgCl2 and Na2EDTA if Mg2EDTA is unavailable: <br />- Combine equal parts of 0.125 M Tris-base, 0.1 M Na2-EDTA dihydrate, 0.2 M MgCl2 hexahydrate, and 0.2 M ascorbic acid, adjust with 10 N NaOH to reach a pH of 6-7.<br />&nbsp; &nbsp; - 0.125 M Tris-base prepared by dissolving 0.151 g Tris-base in 10 mL ultrapure water <br />&nbsp; &nbsp; - 0.1 M Na2-EDTA dehydrate prepared by dissolving 0.372 g Na2- EDTA dehydrate in 10 mL ultrapure water <br />&nbsp; &nbsp; - 0.2 M MgCl2 hexahydrate prepared by dissolving 0.407 g MgCl2 hexahydrate in 10 mL ultrapure water <br />&nbsp; &nbsp; - 0.2 M ascorbic acid prepared by dissolving 0.352 g ascorbic acid in 10 mL ultrapure water

[STEPS]
?. 
?.
TITLE: General initiation protocol for HEK-Blue cells
AUTHORS: Andreas Sagen

[DESCRIPTION]
HEK-Blue is a product from Invivogen, which provide reporter cells for endotoxin-testing among others. Here is a generalized protocol for initial seeding of cells with reporter characteristics.

[BEFORE_START]
Prepare complete initiation medium

[GUIDELINES]
The protocol has been proven to work with different HEK-Blue cells, but not all. Take time to optimize.

[STEPS]
SECTION: Complete initiation medium (CIM)
1. Initially, we want to expand the cells quickly and make them healthy. This require double the amount of FBS and no selection antibiotics in the medium for the first 2-3 passages.
SECTION: Complete initiation medium (CIM)
1.1. In 400 mL DMEM, mix 100 mL FBS

 

Materials:
 
 
SECTION: Complete initiation medium (CIM)
1.2. Aliquot CIM in 50 mL units and store frozen at -20 °C

 

SECTION: Thawing from frozen stock
2. The cells are fragile in the beginning. Take extra good care of the cells when thawing.
SECTION: Subculture of early culture
3. The cells are fragile the first 2-3 passages. To maximize growth, it is important to use a special medium formulation and take extra care when working.
SECTION: Thawing from frozen stock
2.1. Add 11 mL warm CIM in a T-75 flask and incubate for 30 min in a 5% CO2 incubator
SECTION: Thawing from frozen stock
2.3. Transfer vial content to a centrifuge tube with 5 mL warm CIM
SECTION: Thawing from frozen stock
2.2. Get a vial of HEK-Blue cells, and thaw in a warm water bath for approx. 2 min
SECTION: Thawing from frozen stock
2.5. Discard supernatant and resuspend pellet in 1 mL warm CIM
SECTION: Thawing from frozen stock
2.4. Centrifuge with 200 rcf, 5 min
SECTION: Thawing from frozen stock
2.6. Add all content from centrifugation tube to T-75 and incubate in a 5% CO2 incubator

 

SECTION: Subculture of early culture
3.2. Remove supernatant from T-75 and wash with PBS

Materials:
 
SECTION: Subculture of early culture
3.3. Add 3 mL PBS and incubate for 5 min
SECTION: Subculture of early culture
3.4. Add 3 mL CIM and transfer content to centrifugation tube
SECTION: Subculture of early culture
3.5. Centrifuge with 200 rcf, 5 min
SECTION: Subculture of early culture
3.7. Split between 1:2 to 1:6 of resuspended suspension from centrifugation tube to T-75 and incubate in a 5% CO2 incubator

 

Repeat this procedure once, then change to the general subculture protocol
 

SECTION: Subculture of early culture
3.6. Discard supernatant and resuspend pellet in 1 mL warm CIM
SECTION: Subculture of early culture
3.1. Add 11 mL warm CIM in a T-75 flask and incubate for 30 min in a 5% CO2 incubator
TITLE: Soil Sample (Laboratory, English)
AUTHORS: Hsin-Mao   Wu

[DESCRIPTION]
This protocol is for 1 sample site

[GUIDELINES]
This protocol is for 1 sample site

[STEPS]
SECTION: Soil sampling
1. Sample sites have 3 different types
 

SECTION: Soil sampling
2. In one sampling site, choose 5 places as the average
SECTION: Soil sampling
3. Remove the topsoil, take the soil at a distance of 5-7 cm from the soil surface, and put it into a plastic zipper bag
SECTION: Soil sampling
4. Mix the soil evenly, and then add soil into 2 separate HDPE plastic bottles
 

SECTION: Soil sampling
5. Put the “Cold” one into the insulated bag; put the “RT” into a backpack
SECTION: Soil sampling
6. Take 2 pictures:
TITLE:     Direct ELISA for investigating the binding of peroxidase-labeled anti-chicken IgY conjugate with avian immunoglobulins
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The peroxidase-labeled anti-chicken IgY conjugate cross-reacts with many IgY present in the egg of many and diverse avian species.  </div><div class = "text-block">References</div><div class = "text-block">1. Justiz Vaillant AA, Ramirez N, Cadiz A, Ferrer B, Akpaka P, et al. (2013) Separation and Reactivity of Avian Immunoglobulin Y. J Chromat Separation Techniq 4: 173. doi:10.4172/2157-7064.1000173</div></div>

[STEPS]
?. This ELISA is used to study the interaction of anti-chicken IgY-HRP conjugate with diverse avian immunoglobulins.
?. The 96 well microtitre plate is coated overnight at 4°C with 1 µg/µl per well of purified avian immunoglobulins or 50 µl of water soluble fraction from egg yolks of avian species in carbonate-bicarbonate buffer pH 9.6.
?. Then plate is treated with bovine serum albumin solution and washed 4X with PBS-Tween.
?. Then 50 µl of peroxidase-labeled-anti-chicken IgY conjugate diluted 1:15000 in PBS-non-fat milk is added to each well and  incubated for 1.30h at RT. After that the plate is washed 4X with PBS-Tween.
?. Pipette 50 μl of 3,3',5,5' - tetramethylbenzidine (TMB; Sigma-Aldrich) to each well.
?. The reaction is stopped with 50 µl of 3M H2SO4 solution.
?. The plate is visually assessed for the development of colour and read in a microplate reader at 450 nm.
?. A cut-off point should be calculated as the mean of the optical density of negative controls x 2 SD.
TITLE: Cell seeding
AUTHORS: PMAT0001 

[STEPS]
?. Take cells from the freezer and thaw for   in the  water bath.
37 °C
?. Take a T75 plate and place all needed items in ethanol-sterilized laminar flow hood.
?. Draw the needed amount of cells into a 50mL centrifuge tube.
?. Add about  of PBS into the centrifuge tube.
7 mL
?. Spin the mixture in a centrifuge for   at 1500rpm.
?. Decant supernatant  and add PBS to the cell pellet once again.
?. Resuspend cell pellet with the PBS using a micropipette.
?. Repeat steps 5 to 7.
?. Add DMEM (with FBS and antibiotics) into centrifuge tube and resuspend cell pellet.
?. Add DMEM to the T75 plate (sufficient to cover the entire surface area, about  ).
5 mL
?. Add   of the cell mixture into the T75 flask.
5 mL
?. Incuabte T75 flask at , 5% CO2.
37 °C
TITLE: Veiled Chameleons (Chamaeleo calyptratus) 2021 Environmental Summary, Reptile &amp; Aquatics, Stowers Institute for Medical Research
AUTHORS: Diana P Baumann, Richard Kupronis

[DESCRIPTION]
Veiled Chameleons (Chamaeleo calyptratus) are an increasingly popular model organism, and we have maintained a colony at the Stowers Institute since 2010. Due to the poikilothermic nature of these animals, achieving the correct macro and microenvironments plays an important role in successful long term colony maintenance. Despite being bred in captivity for many generations, relatively little husbandry information has been published regarding housing in a research environment. Recognizing that husbandry conditions and environmental parameters can impact research results we provide information about the housing, husbandry, maintenance, nutrition, and health for our colony of Veiled Chameleons. This information will be applicable to any chameleon housed in the facility in 2021.

[STEPS]
TITLE: 03 Ligation
AUTHORS: TJUSLS China

[STEPS]
?. Mix together:5×T4 DNA Ligase Buffer      2μlT4 DNA Ligase                      0.5—1μlVector                                     as requiredInsert                                       as requiredddH2O                                     Add to 10μl
?. Heat in water bath or heat block at 22℃ for 30min.
22 °C
TITLE: Super Slim Keto Gummy Bears (Scam Exposed 2022) - Is Really worth Buying?
AUTHORS: H  H

[DESCRIPTION]
This extraordinary keto equation centers around not one, however three types of BHB ketones, so you can get a definitive thinning mix. Inside this blend, you can track down magnesium, sodium, and calcium BHBs to get the best supplements for your body. With these strong ketones, you can intensify the rate that the keto diet transforms muscle versus fat into usable energy.

[STEPS]