eamag/protocols-with-steps huggingface dataset:
TITLE: In vitro transcription of guide RNAs and 5&#39;-triphosphate removal
AUTHORS: Moritz F Schlapansky, Eric Aird, Mark Dewitt,  Julia Wong, Beeke Wienert

[DESCRIPTION]
sgRNA template assembly, in vitro T7 transcription, and sgRNA column cleanup to remove 5'-triphosphate groups

[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.
Assembly Oligos:
T7FwdVar oligo (5’-TAATACGACTCACTATAG--protospacer sequence—GTTTCAGAGCTATGCTGGAAAC-3’ )
T7RevLong oligo (5’-AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTCAACTTGCTATGCTGTTTCCAGCATAGCTCTGA-3’ )

Amplification Primers:
T7FwdAmp primer (5’-TAATACGACTCACTATAG-3’)
T7RevAmp primer (5’-AAAAAAAGCACCGACTCGGTGC-3’ )
 

Designing the guide sequence: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.
Making the template: we have used the Phusion-HF DNA polymerase. Although the amplification primers have quite different Tm’s, they work together fine. 



[STEPS]
1.7. Move spin column to an RNAse-free 1.5 ml microfuge tube
Add 33 µl DEPC-treated H2O; spin 1 min
Optional: Repeat the elution to collect any remaining RNA on the column and increase RNA concentration. 
1.6. Move spin column to a new collection tube and spin for 1 min at 10.000 g to dry the membrane completely.

 
1.5. Add 500 µl RPE Buffer; Spin for 2 min at 10.000 g.
1.4.  Add 700 µl RWT Buffer; Spin for 15 sec at 10.000 g.
 Discard the flow-through.

1.3. Transfer sample (~900 µl) to an RNeasy mini spin column; spin for 15 sec at 10.000 g.
Discard the flow-through.
 

1.2. Add 450 µl Isopropanol and mix well by pipetting.
 

1.1. Adjust sgRNA sample to a volume of 100 µl with RNase-free water.
Add 350 µl RLT Buffer to the sample and mix well by pipetting
1. sgRNAs need to be purfied before dephosphorylation and transfection. There are different methods one could purify their sgRNAs. We therefore tested different purification kits and found that the Qiagen miRNeasy Tissue/Cells Advanced Mini Kit gives the most consistent and highest yields. To purify our sgRNAs with this kit, we follow the manufacturer's instructions with the following modifications:
TITLE: Open Field test to assess spontaneous locomotion behavior in parkinsonian mice
AUTHORS: natalia.lopezgonzalezdelrey, Zachary Gaertner

[DESCRIPTION]
The Open Field task is a simple sensorimotor test used to determine general activity levels, gross locomotor activity, and exploration habits in rodent models of CNS disorders.

[STEPS]
SECTION: Open Field Test
1. Total duration: 1 day
SECTION: Protocol
2. Place the animal cage in the testing room to allow the animals to acclimate. 60 min 
SECTION: Protocol
3. Place the animal in the testing boxes. The animals are transferred to the center of a 40cm by 40cm box in the quiet dark room.
SECTION: Protocol
4. Video record and record freely moving behavior in the testing cage.5 min 

SECTION: Protocol
5. Return the mouse to its cage.
SECTION: Analysis
6. The footage is then analyzed by an automated tracking system for the following parameters: distance moved, velocity, and time spent in pre-defined zones.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol describes how to make an acoustic droplet ejection compatible plate for liquid handling on the Echo 555 using an epMotion 5075. The protocol expects (4) 96-well primer plates and an empty 384-well low dead volume (LDV) echo plate. The 4 plates will be compressed in a fully interweaved layout. The following images depict this pattern. 
  

 





[BEFORE_START]
Please wear at least the minimum required personal protective equipment.
Ensure that all necessary kit components are available as well as user-supplied consumables.
Remove nuclease and nucleotide contamination from work surfaces and instruments prior to starting using an appropriate solution, such as RNase AWAY™ (Thermo Scientific™ catalogue: 700511), followed by wiping with 70% to 100% molecular biology grade ethanol to remove additional contaminants.

[STEPS]
SECTION: Prepare primer plates
?. 
SECTION: Prepare primer plates
?. 
SECTION: Setup epMotion automation platform
?. 
SECTION: Execute automated protocol
?.
TITLE: GenomeTrakr WGS Protocol Collection and Workflow for MiSeq
AUTHORS: Tina.Pfefer  , Julie  Haendiges, Maria   Balkey, Ruth  Timme

[DESCRIPTION]
Here we have created a collection of all the protocols used for WGS using the MiSeq, in order, from sample extraction to NCBI submission. 

This collection has three sections:

Section 1: WGS Wet lab workflow for Illumina MiSeq
Section 2: Dry lab workflow for sequence QC and NCBI submission - Direct Submission
Section 3: Dry lab workflow for sequence QC and NCBI submission - PulseNet labs


Associated protocols:

Querying the NCBI database for GenomeTrakr data (protocols.io)
NCBI data curation protocol - SOP for editing GenomeTrakr submissions (protocols.io)


[STEPS]
SECTION: Wet lab
2. DNA Extraction
 

SECTION: Wet lab
3. DNA Quantification
 

SECTION: Wet lab
4. Library Preparation
 

SECTION: Wet lab
5. Sequencing
 

SECTION: Dry lab - Direct Submission
7. Check sequence quality:
 


SECTION: Dry lab - Direct Submission
6. Dry lab workflow for sequence QC and NCBI submission - Direct Submission: 


The following protocols are also included in a Springer Methods book chapter collection: Utilizing the Public GenomeTrakr Database for Foodborne Pathogen Traceback (protocols.io)

SECTION: Dry lab - Direct Submission
8. Populate BioSample AND SRA metadata templates:
 

SECTION: Dry lab - Direct Submission
9. Submit sequence and metadata to NCBI:
 

SECTION: Wet lab
1. WGS Wet lab workflow for Illumina MiSeq
SECTION: Dry lab - PulseNet submission
11. Dry lab workflow for sequence QC and NCBI submission - PulseNet labs:
SECTION: Dry lab - PulseNet submission
12. Check sequence quality:

Follow the CDC's guidance for assessing QC in BioNumerics
SECTION: Dry lab - PulseNet submission
13. Populate NCBI template: 
 

SECTION: Dry lab - PulseNet submission
14. Submit sequence and metadata to NCBI:

Follow the CDC's guidance for NCBI submissions through BioNumerics.
SECTION: Dry lab - PulseNet submission
15. Update, retract, or replace these records in NCBI databases, if necessary:

NCBI data curation protocol - SOP for editing GenomeTrakr submissions (protocols.io)





SECTION: Dry lab - Direct Submission
10. Update, retract, or replace these records in NCBI databases, if necessary:

NCBI data curation protocol - SOP for editing GenomeTrakr submissions (protocols.io)
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
The following protocol describes the acoustic droplet ejection dispenses needed for a minitaturized 16S PCR reaction using the Echo 550. The protocol expects (1) 384-Well 16S Illumina Primer Plate in an echo qualified Low Dead Volume (LDV) plate, (1) 384-Well Extracted gDNA in an echo qualified Polypropylene (PP) plate, and (3) 384-Well PCR Plates in twin.tec PCR plates. 

[BEFORE_START]
Please wear at least the minimum required personal protective equipment.
Ensure that all necessary kit components are available as well as user-supplied consumables.
Remove nuclease and nucleotide contamination from work surfaces and instruments prior to starting using an appropriate solution, such as RNase AWAY™ (Thermo Scientific™ catalogue: 700511), followed by wiping with 70% to 100% molecular biology grade ethanol to remove additional contaminants.

[STEPS]
SECTION: Prepare Plates
?. 
SECTION: Prepare Plates
?. 
SECTION: Execute acoustic droplet ejection transfers
?. 
SECTION: Execute acoustic droplet ejection transfers
?. 
SECTION: Seal plates
?.
TITLE: Q5® Site-Directed Mutagenesis Kit Quick Protocol (E0554)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
This is the quick protocol for the Q5® Site-Directed Mutagenesis Kit (E0554).

[BEFORE_START]
Primers should be designed with 5´ ends annealing back-to-back. We recommend using the NEB online design software, NEBaseChanger™.

[STEPS]
SECTION: Exponential Amplification (PCR)
1. Assemble the following reagents in a thin-walled PCR tube.
 

SECTION: Exponential Amplification (PCR)
2. Mix reagents completely.
SECTION: Exponential Amplification (PCR)
3. Transfer to a thermalcycler and perform the following cycling conditions:
 

 
SECTION: Kinase, Ligase & DpnI (KLD) Treatment
4. Assemble the following reagents:
 

SECTION: Kinase, Ligase & DpnI (KLD) Treatment
5. Mix well by pipetting up and down.
SECTION: Kinase, Ligase & DpnI (KLD) Treatment
6. Incubate at Room temperature for 5 min.
SECTION: Transformation
7. Add 5 µL from previous step to 50 µL.
SECTION: Transformation
8. Incubate on ice for 30 min. 
SECTION: Transformation
9. Heat shock at 42 °C for 30 s. 
SECTION: Transformation
10. Incubate on ice for 5 min. 
SECTION: Transformation
11. Add 950 µL. 
SECTION: Transformation
12. Gently shake at 37 °C for 60 min.
SECTION: Transformation
13. Spread 40 µL–100 µL onto appropriate selection plate.
SECTION: Transformation
14. Incubate 60 min at 37 °C.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="font-weight: 400;">This protocol describes the production of a reference-quality <em>de novo</em> transcriptome assembly for the spiny mouse (<em>Acomys cahirinus</em>).</span><span style="font-weight: 400;"> These methods can be applied to other RNA-Seq datasets to generate high-quality transcriptome assemblies in other species. V</span>alidation and description of assembly output is described in 'De novo transcriptome assembly for the spiny mouse (<em>Acomys cahirinus</em>)' <em>bioRxiv</em>, p.076067.</p>

[STEPS]
?. 
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TITLE: Immunohistochemistry for CD8+ staining in Breast Cancer Tissue
AUTHORS: Freda Halim

[DESCRIPTION]
These are protocols used for study of CD8+ Cell Count in Luminal B Her-2 negative BC patients. We used using paraffin sections of 66 samples and stained the slides for CD8+ antibody, using primary antibody CD8 (clone C8/144B, dilution 1:100; Dako

[STEPS]
SECTION: Deparaffinization and rehydration
1. Incubate slides in Xylenes for 3 minutes          
SECTION: Deparaffinization and rehydration
3. Rinse with running tap water and aquadest
SECTION: Deparaffinization and rehydration
2. Rehydrate slides in 100% Ethanol, 96% Ethanol, 70% Ethanol each for 3 minutes
SECTION: Blockage of Endogenous Peroxidase
4. Incubate slides in 3% H202 for 15 minutes
SECTION: Blockage of Endogenous Peroxidase
5. Rinse with running tap water and aquadest
SECTION: Antigen Retrieval
6. Antigen Retrieval with Tris EDTA ( pH9) with pressure cooker, in 95 degree Celcius temperature
SECTION: Antigen Retrieval
7. Open the lid and cool down in room temperature 
SECTION: Antigen Retrieval
8. Rinse slides with running water and aquadest for 5 minutes
SECTION: Antigen Retrieval
9. Rinse in PBS ( Phosphate Buffer Saline ) in pH 7.40-7.60
SECTION: Antigen Retrieval
10. Excell Block
SECTION: Antigen Retrieval
11. Rinse in PBS in pH 7.40-7.60
SECTION: Primary Antibody
12. Wipe excess liquid around the tissue, apply primary antibody :CD8 (clone C8/144B, dilution 1:100; Dako
SECTION: Primary Antibody
13. Incubate for 60 minutes and then rinse with PBS for 5 minutes
SECTION: Secondary Antibody
14. Apply Excell Link as secondary antibody for 15 minutes, then rinse with PBS for 5 minutes
SECTION: Secondary Antibody
15. Apply Excell HRP as secondary antibody
SECTION: Signal Detection/Histochemistry
16. Apply DAB ( Diamino-benzidine ) 80-100μL for 10 minutes , Rinse with running tap water and aquadest for 5 minutes
SECTION: Signal Detection/Histochemistry
17. Apply Hematoxylline for 1 minutes, Rinse with running tap water and aquadest for 5 minutes
SECTION: Signal Detection/Histochemistry
18. Apply Tatcha's bluing solution and rinse with running tap water and aquadest for 5 minutes
SECTION: Dehydration and Clearing
19. Clear excess water from slides and Dehydrate Slides in 70%, 96%, 100% for 5 minutes each
SECTION: Dehydration and Clearing
20. Incubate slides in Xylenes for 5 minutes
SECTION: Dehydration and Clearing
21. Mount the slides
TITLE: Parse Evercode Fixation Protocol v2.0.1 for cells or nuclei -- University of Minnesota TMCs
AUTHORS: Parse Biosciences, Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
https://www.parsebiosciences.com/products/evercode-whole-transcriptome

Fixation for Parse Evercode sc/snRNAseq 
Includes details for both cells and nuclei fixation -- However, each require a different product kit from Parse to complete.  
Please check for the most recent version of the protocol before going forward. 

[STEPS]
1.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is used for performing Type IIS assembly by either <em>BsaI</em> or <em>SapI</em>-mediated restriction/ligation using the Loop assembly system.</p>
<p> </p>
<p>Loop assembly comprises 8 receiver plasmids in odd and even levels (4 per level), which contain directional overhangs for higher level assembly. Odd and even level receiver plasmids are used in a recursive schema for assembly in iterative 'loops'. Any assembly (except for L0 part composition into a L1 transcriptional unit) requires the usage of 4 donor plasmids (each in a specific position) and one receiver plasmid. <em>BsaI</em> is used for assembly into odd levels using kanamycin selection, and <em>SapI</em> is used in even levels using spectynomycin selection. Loop odd receivers use the Common Syntax standard for L0 part assembly into TUs (Patron, <em>et al.</em> 2015 [10.1111/nph.13532]). Then, 4 L1 plasmids (positions 1-4) are assembled into an even receiver plasmid to provide a L2 assembly (4 TUs). For higher level assemblies the same assembly structure is followed, 4 L2 plasmids (positions 1-4) with an odd receiver to yield a L3 assembly (16 TUs).</p>
<p> </p>
<p>The Loop type IIS assembly protocol is based on: </p>
<p class="p1"><span class="s1">“Patron, NJ (2016) DNA Assembly for Plant Biology. Current Protocols in Plant Biology 1:1-13 [doi: 10.1002/cppb.20038]”, but with slight modifications for DNA concentrations:</span></p>
<p> </p>
<p><strong>This protocol uses a target amount of each donor DNA of 15 fmol and 7.5 fmol for receiver plasmid DNA in a 10 µL reaction. In order to perform accurate dispensing, the plasmid DNA is diluted to their corresponding concentration of 15 fmol/µL for donor parts and of 7.5 fmol/µL for the receiver plasmid, and then 1 µL of each plasmid is added to the DNA mix. The plasmid part target concentration (in ng/uL) equals to each donor plasmid length / 100, and of the receiver plasmid length / 200. </strong></p>
<p> </p>

[BEFORE_START]
<p>Thaw 10X T4 ligase buffer at RT and then leave on ice. Keep BSA on ice. Briefly vortex 10X ligase buffer before using.</p>

[GUIDELINES]
<p>Each level (odd and even) contains four 'receiver' plasmids:</p>
<p> </p>
<p>pOdd-1, 2, 3, 4</p>
<p>pEven-1, 2, 3, 4</p>
<p> </p>
<p>For a the assembly of L0 parts into a L1 TU, a variable number of parts can be used for the asssembly of the TU, however, for higher level assemblies only 4 donor modules can be assembled into any receiver.</p>

[STEPS]
?. 
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?.
TITLE: Sample preparation
AUTHORS: philippe.bechtold 

[STEPS]
?. [Sample Preparation]
Take the swab (NP or OP)
?. [Sample Preparation]
Insert swab into Tube H with lysis buffer ( , consisiting ofand -)
500 µl
[violent shaking]
[Incubation]
?. [Sample Preparation]
Dispose swab and  add   and
[EtOH]
[Magnetic beads]
?. [Sample Preparation]
Discard supernatant after pulling the particles to the side
?. [Washing 1]
Add  , resuspend
[EtOH]
?. [Washing 1]
Discard supernatant after pulling the particles to the side
?. [Washing 2]
Add  , resuspend
[EtOH]
?. [Washing 2]
Discard supernatant after pulling the particles to the side
?. [Elution]
Add  , resuspend
[RNAse free water]
?. [Elution]
Pull the particles to the side and take supernatant to load the chip as described
TITLE: Single cell dissociation of brain organoids
AUTHORS: michela.deleidi, María José Pérez J.

[DESCRIPTION]
This protocol details about single cell dissociation of brain organoids.

[STEPS]
SECTION: Single cell dissociation of brain organoids
1. Mix 500 µL DNAse with 5 mL Papain. 
 



SECTION: Single cell dissociation of brain organoids
2. Transfer single or pooled organoid to 60 mm dish.
SECTION: Single cell dissociation of brain organoids
3. Aspirate excess media, add 2.5 mL Papain + DNAse solution.

SECTION: Single cell dissociation of brain organoids
4. With a razor blade mince organoid (<1 mm).
SECTION: Single cell dissociation of brain organoids
5. Transfer plate to an orbital shaker   (inside incubator).

SECTION: Single cell dissociation of brain organoids
6. With 1-mL pipette dissociate pieces (Mix up-down 30 times).
SECTION: Single cell dissociation of brain organoids
7. Put in orbital shaker 20 min.

SECTION: Single cell dissociation of brain organoids
8. In the meantime, add 5 mL Earle´s medium + 3 mL Inhibitor to a 15-mL conical tube.

SECTION: Single cell dissociation of brain organoids
9. Remove samples from the orbital shaker. With a 1-mL tip, mix up-down 30 times.
SECTION: Single cell dissociation of brain organoids
10. Take 2 mL (upper part) into new tube using a 40 µm cell strainer. Wait 1-3 min to debris to settle.

SECTION: Single cell dissociation of brain organoids
11. Transfer cell suspension to the inhibitor tube. Invert to mix 5 times.
SECTION: Single cell dissociation of brain organoids
12. Centrifuge 300 rpm, 7 min.

SECTION: Single cell dissociation of brain organoids
13. Aspirate supernatant, resuspend in 500 µL to 1 mL 0.5% BSA-PBS (Up-down 30 times).

SECTION: Single cell dissociation of brain organoids
14. Filter the resuspended cells (900 µL) with a 30 µm cell strainer.

SECTION: Single cell dissociation of brain organoids
15. Count the cells for the final suspension and dilute. Resuspend at 1000 cells/μl in 0.04% BSA-PBS.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is the protocol to extract RNA from Sterivex filters from RNA-SIP experiments carried out with seawater, vent fluids, etc.</p>

[STEPS]
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TITLE: Imaging - Stanford TMC
AUTHORS: John Hickey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>See our detailed protocol published with the following title: </span><span style = "font-weight:bold;">CODEX multiplexed tissue imaging with DNA-conjugated antibodies.</span></div></div>

[STEPS]
?. See our detailed protocol published with the following title: CODEX multiplexed tissue imaging with DNA-conjugated antibodies.
TITLE: Worm Freezing Buffer
AUTHORS: Adrien   Assie, Buck  Samuel

[DESCRIPTION]
Worm Freezing buffer recipe according to Brenner, S. (1974). Genetics77, 71. Found on wormbook see external link

[STEPS]
1. 150 mLof 100 % volume glycerol


2. 25 mL of 1 Molarity (M) Potassium Phosphate Buffer, pH 6.0


3. 2.85 gof NaCl

4. 500 mLof M9 Buffer




5. Adjust deionized water to1000 mL

6. Autoclave
TITLE: Installation instructions for RNA-seq analysis using a conda environment
AUTHORS: Laise Moraes

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of this protocol is to define and provide instructions for creating a conda environment for phylogenetic analysis</div></div>

[STEPS]
?. [Installing Miniconda]
Create a directory called softwares to your HOME directory, switch to it and then download the 64-bit Python 3 Miniconda installer.Install Miniconda quietly, accepting defaults.After installation, remove the Miniconda installer from directory.Set the Miniconda permanent PATH and update conda packages to the latest compatible version.
?. [Creating conda environment]
Create a YML file to build a phylogenetic analysis environment called rna-seq.
?. [Creating conda environment]
Create the environment from the phy.yml file
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div title="Page 54">
<div>
<div>
<p>Platelet activation was quantitatively assessed on a randomly selected subgroup of 10 patients (not treated with acetylsalicylic acid) in un-processed blood by the PACT (Platelet activation test, adjusted from Roest 2013). In the original paper, Roest performs the tests at room temperature while in this manuscript, the PACT test is performed at 37°C which requires higher agonist doses. The PACT is based on platelet activation induced by addition of a specific agonist to whole blood and gives specific insight in the granule release capacity and in the aggregation potential of platelets. The test contains three agonists to activate the platelets: (1) the protease activated receptor (PAR-1) agonist thrombin receptor activator peptide (TRAP f.c. 30 μM, SFLLRN, H-2936; Bachem, Germany), (2) the glycoprotein VI (GPVI) agonist collagen-related peptide (CRP f.c. 5 μg/ml, Pro- fessor Farndale, university of Cambridge, UK), and (3) the P2Y12 agonist ADP (f.c. 30 μM, 01897, Sigma-Aldrich, Zwijndrecht, the Netherlands) in HEPES-buffered saline (HBS, 10 mmol/L HEPES, 150 mmol/L NaCl, 1 mmol/L MgSO4, 5 mmol/L KCL, pH 7.4). The reaction mixtures also contain three antibodies directed against GPIb (APC-conjugated CD42b, BD Bioscience), activated αIIbβ3 (FITC-conjugated PAC-1) and P-selectin (PE-conjugated CD62P) purchased from BD Pharmingen (Franklin Lakes, USA). Whole blood was heated at 37°C for 10 min and the tests were performed at 37°C. Whole blood was diluted 1:4 in HEPES-buffered saline and 5 μl of this diluted blood was added to each reaction mixture. Reactions were stopped by adding 250 μl fixation solution (137 mmol/L NaCl, 2.7 mmol/L KCl, 1.12 mmol/L NaH2PO4, 1.15 mmol/L KH2PO4, 10.2 mmol/L Na2HPO4, 4 mmol/L EDTA, 0.5% formaldehyde) after exactly 20 min of incubation at 37°C. Flow cytometry was used to distinguish between platelets and other cells on forward and sideward scatter pattern and by gating on the CD42b positive cells. Fluorescent intensity in the FITC gate and PE gate was selected to determine activated αIIbβ3 and P-selectin density, respectively, and results are expressed as median fluorescent intensity (MFI).</p>
</div>
</div>
</div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
How to Make a 0.5M TCEP Stock Solution

[GUIDELINES]
<a href="http://store.p212121.com/TCEP-HCl/" target="_blank">TCEP</a> can be dissolved in many types of aqueous buffers, and at a wide range of pH levels and still maintain stability<br /><br />This allows you to prepare TCEP working concentrations and 10X stock solutions in the buffer of your choice before use.While TCEP can be used in Phosphate buffers, it has the tendency to be unstable around neutral pH levels.<br /><br />If the TCEP is needed in the buffer, prepare it immediately before use.<br /><br />A 50 mM TCEP solution may be substituted for <a href="http://store.p212121.com/DTT/">DTT</a> or &beta;-Mercaptoethanol (&beta;-ME) in SDS-PAGE loading buffers. TCEP cannot be used for isoelectric focusing, due to its charge in solution.

[STEPS]
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TITLE: Cardiologist
AUTHORS: Dr rAKathy Kathy rAKathy. Davis, Kathy  Mach

[DESCRIPTION]
Heart failure monitor decision diagnosed medical condition needing medication results heart attack know further then artery blocking the valve stopping blood flow to the cause heart attack. considered as heart failure monitor access proper way to make decision.second opinion could be option if not comfortable with heart failure diagnosed medical condition of EKG complaint of emergency further examine with high blood pressure cholesterol levels  results of lab work basic level high pressure cholesterol extremely high  above normal range pressure unstable condition heart attack smoking causing different type of reaction of health condition drinking alcohol level basic witn test abnormal specimen was identify causing pain with the substance but wasn't aware of heart attack upon visit to emergency room with alcohol level . 

[STEPS]