eamag/protocols-with-steps huggingface dataset:
TITLE: BSCI:414 Lab 1--Benchling and PCR
AUTHORS: Harley King

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Lab 1 Overview</div><div class = "text-block">	Discuss and Begin Lab Notebooks in Benchling</div><div class = "text-block">	Import and Explore DNA in Benchling</div><div class = "text-block">	Make primers in Benchling</div></div>

[STEPS]
?. [Benchling Lab Notebook Tutorial]
Introduce last semester's lab notebooks.
?. [Benchling Lab Notebook Tutorial]
Discuss features of a good lab notebook e.g. dates, navigation.
?. [Students Begin Lab Notebook]
Sign into Benchling, join and find project "USG BSCI:414 Fall 2020 Lab".
?. [Students Begin Lab Notebook]
Begin your own lab notebook by:Creating folder entitled "FirstName LastNameLab Notebook" under Projects/USG BSCI:414 Fall 2020 Lab/"Clicking "+" sign and "Entry>Blank Entry". Entitle this the same as the folder e.g. Harley King Lab Notebook"Begin by including a link to this protocol.
?. [Benchling DNA Tutorial]
Overview of Benchling sequencesImporting DNACopying/pasting DNAUnderstanding DNA featuresUndo history
?. [Benchling DNA Tutorial]
Students Import DNA sequencesCreate a new folder called "FirstName LastName Lab 1" under "Projects/USG BSCI:414 Fall 2020 Lab/Lab 1"Import a DNA sequence and attempt the following:Delete part of the DNA. Undo the deletion.Add bases to the DNA.Delete the additional bases.Do crazy stuff and then use the "Undo History" to reset the plasmid back to its original state.
?. [Create and Run Virtual PCR Tutorial]
Import DNA sequenceMake primers
?. [Students Create and Run Virtual PCR]
Into your lab 1 folder, copy the sequence "Addgene_SARS_Spike_pGBW-m4136458" from "Projects/USG BSCI:414 Fall 2020 Lab/". Put your name in front of the sequence e.g. "HarleyKingcopy_Addgene_SARS_Spike_pGBW-m4136458".Select a sequence from this plasmid Select primer icon from right side then "CREATE PRIMERS" then "Wizard".The target range can be any shift-selected bp region or you can enter numbers like 0 to 3000. click "Generate Primers".With luck, a few primer options will appear on the next screen. Notce that each contains a forward "FWD" and reverse "REV" primer. Both are necessary for successful PCR. Select one "FWD" and one "REV" primer with a checkmark. Select "Save Selected Primers". In the box that pops up, save them to your Lab 1 folder. Return to "Sequence Map". Scroll through the sequence and find your primers. Rename the primers by right clicking>Edit and changing the name. Shift select between the primers. How many bp is the fragment? Note this in your lab notebook.
?. [Update Lab Notebooks]
In your lab notebook, use "@ mention" to link the name of your plasmid. Also link the renamed primers. Note how many bp your PCR fragment is.
TITLE: Collection of SOPs for Covid-19 Shutdown Re-Entry, Srivastava Lab
AUTHORS: Srivastava Lab

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Disclaimer</span><span>: The SOPs presented have been designed by the Srivastava Lab at Harvard University and are being shared outside of Harvard for informational purposes only. If you choose to reuse or repurpose these SOPs in another location, please note that you do so at your own risk; you should ensure that any local guidance is also adhered to. None of the authors, contributors, administrators, or anyone else associated with </span><a href="http://protocols.io" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">protocols.io</span></a><span>, can be held responsible for your use of the information contained in or linked to from these web pages.</span></div></div>

[STEPS]
TITLE: Exploring the components of feedback facilitation: A systematic review
AUTHORS: Michael Sykes, Sarah Alderson, Owen Thomas, Karen Caulfield, Lisa Robinson, Matthew Quigley, Lavanya Gupta, Zahava Rosenberg-Yunger

[DESCRIPTION]
Audit and feedback is a much-used intervention that involves the delivery of feedback on performance over a specific period of time. Delivering feedback facilitation as a co-intervention alongside audit and feedback may enhance the response to feedback. The content and delivery of feedback facilitation may influence its effectiveness. The primary aim of this systematic review is to describe the content and delivery of feedback facilitation co-interventions used in trials of audit and feedback. We will include studies delivering feedback facilitation alongside audit and feedback, as determined by the Cochrane review of audit and feedback. We will extract data from papers describing the trial, from publicly available protocols and from companion papers. We will describe the content and delivery of feedback facilitation using an adapted TIDieR framework (Hoffmann et al 2014). Each paper will be reviewed by 2 reviewers; the reviewers will extract separately, and disagreements will be resolved through discussion. Data will be recorded in Excel. We will use thematic analysis to describe the intervention. If possible, we will describe the effectiveness of similar feedback facilitation interventions graphically. 

[STEPS]
TITLE: RNA isolation
AUTHORS: Angkana Kidtiwong

[DESCRIPTION]
RNA isolation for sample stored in TRIzol reagent

[STEPS]
SECTION: RNA isolation
1.      
 
Prepare the centrifuge at 4°C and meanwhile thaw the frozen sample (in TRIzol, stored at -80°C) at room temperature for about 10-15 minutes.


SECTION: RNA isolation
2. Add chloroform at 20% of the sample volume to the sample tube (typically, 1 ml of TRIzol sample, so add 200 µl of chloroform in sample tube) and incubate at room temperature for 5 minutes. Mixing from time to time during the incubation
SECTION: RNA isolation
3. Centrifuge at 13,000 rpm, 4°C for 15 minutes.

 


SECTION: RNA isolation
3.1. In a meanwhile, prepare the mixture of RNA precipitation as follow:
a. Isopropanol: 0.8 times the total volume of supernatant plus 400 µl DEPC water (typically, take 400 µl of supernatant after centrifugation (clear part), add 640 µl of isopropanol) per sample.
b. DEPC water: 400 µl per sample.
c. Glycoblue: 1-2 µl per sample.
Note: Always prepare 2-3 extra mixtures.
SECTION: RNA isolation
4. Transfer 400 µl of supernatant after centrifugation (clear part) to a new Eppendorf tube - Carefully pipetting 
SECTION: RNA isolation
5. Add 1,041 µl of the RNA precipitation mixture (400 µl DEPC water + 640 µl isopropanol + 1 µl Glycoblue) to the supernatant. Incubate on ice for 10-30 minutes.
SECTION: RNA isolation
6. Centrifuge at 13,000 rpm, 4°C for 15 minutes.
 

SECTION: RNA isolation
7. Check if a blue pellet has formed at the bottom of the Eppendorf tube, then discard the supernatant and add 1 ml of 75% ethanol (ETOH volume is proportional to TRIzol volume).
SECTION: RNA isolation
8.  Centrifuge at 13,000 rpm, 4°C for 5 minutes.

 

SECTION: RNA isolation
9. Discard the supernatant, then add 1 ml of 75% ethanol and transfer everything to a new Eppendorf tube – Ensure the pellet is transferred as well.
SECTION: RNA isolation
10. Centrifuge at 13,000 rpm, 4°C for 5 minutes.

 


SECTION: RNA isolation
11. Discard all ethanol and air-dry the pellet in the chamber for 5 minutes.
SECTION: RNA isolation
12. Add 20 µl of nuclease-free water to each Eppendorf tube
SECTION: RNA isolation
13. Prepare the RNA treatment mixture (10 µl per sample) of:
a. 6 µl of nuclease-free water.
b. 3 µl of DNase buffer.
c. 1 µl of DNase enzyme.
Note: Always prepare 1-2 extra mixtures.
SECTION: RNA isolation
14. Add 10 µl of the RNA treatment mixture to the sample and incubate at 37°C for 20 minutes on thermo shaker incubator
SECTION: RNA isolation
15. Add 3 µl of DNase inactivation reagent and incubate for 2 minutes at room temperature.
SECTION: RNA isolation
16. Centrifuge at 13,000 rpm, 4°C for 3 minutes.
 

SECTION: RNA isolation
17. Transfer 25 µl of the supernatant to a 1.5 ml Eppendorf tube – keep on ice until RNA measurement.
TITLE: DAB Solutions
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">3, 3´ Diaminobenzidine (DAB) is a commonly used chromogen for histological staining.   In the presence of peroxidase enzyme, DAB produces a brown precipitate that is insoluble in alcohol and xylene.  Peroxidase catalyzes hydrogen peroxide oxidation of substrate DAB. This SOP provides instructions to prepare four different formulations of DAB.  DAB for Immunohistochemical staining (IHC), DAB for Acetylcholinesterase (AChE) Staining, Nickel Enchanced DAB for Biocytin staining, and DAB for Biocytin staining.</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: Bacterial Genomics24
AUTHORS: George Odette

[DESCRIPTION]
This is the protocol for going through the bacterial genomics course.

[STEPS]
SECTION: Installing software for the Bacterial Genomics24
1. We will install many of the software we will use using conda.
Conda
To install conda, run the following script

 
Once the download is complete, run this script:

 
The first time you call conda you might get the error below:
conda: command not found
 If that happens, run this script to add conda to path:

 
But again, if you refresh the shell, you will bump into conda: command not found again. So, add the script to the bashrc file.
Open bashrc using 
 
 The add the export PATH=~/anaconda3/bin:$PATH at the bottom of the file. Click ctrl + o, to save, press enter, the ctrl + x to exit. 
Configuring bioconda
Run the following scripts to configure Bioconda
TITLE: Nf1 loxP site verification
AUTHORS: Greig Couasnay, Florent Elefteriou

[DESCRIPTION]
We have found that our Nf1 flox mouse colonies contained mice that had lost one of the two loxP sites inserted into the Nf1 gene (Parada laboratory, PMID:11297510, JAX# 017639), leading to loss of DNA recombination upon Cre-recombinase activity and loss of previously identified skeletal phenotypes. This is likely to occur in other Nf1 flox mouse colonies.
We describe here a strategy to verify conservation of the loxP sites within the Nf1 allele and to sequence the loxP1 and loxP2 sites for detecting potential mutations or deletions. This may be done if the expression of Nf1 (qPCR) is not reduced in CreTg/+; Nf1 flox/flox tissues of interest.

[STEPS]
SECTION: Nf1 loxP site verification
1.   
Primers for the Nf1  loxP1 site:
P1: 5’-CTTCAGACTGATTGTTGTACCTGA-3’
P4: 5’-TGATTCCCACTTTGTGGTTCTAAG-3’ 
P3: 5'-ACCTCTCTAGCCTCAGGAATGA-3'

Primers for the Nf1 loxP2 site:
LoxP2For: 5’-GCTTTAGCTTCTGGAAATGTGAA-3’ 
LoxP2Rev: 5’-GCGGGCTAAAATGGCAATGTCG-3’  
SECTION: Nf1 loxP site verification
2. gDNA preparation from NaOH lysate
Cut a 2-3mm piece of tail/ear
Add it to 300uL of lysis solution (NaOH 50mM)
Heat for 15 min at 95C
max rpm, 15 min, room Room temperature


SECTION: Nf1 loxP site verification
3. PCR reaction
SECTION: Nf1 loxP site verification
5. 
 

SECTION: Nf1 loxP site verification
3.3.    PCR cycle program
 
 

SECTION: Nf1 loxP site verification
3.1. From a working solution of 10uM, use 1 uL of each primer (200nM each in a total volume of 20uL)
Reaction for loxP1:  P1 + P2 + P3
Reaction for lopP2: LoxP2For + LoxP2Rev
SECTION: Nf1 loxP site verification
3.2. Per reaction, mix 0.5uL of gDNA from the NaOH lysate (from step 2), 1 uL of each primer (from step 3.1), 10 uL   mix, and add water for a total volume of 20 uL 

SECTION: Nf1 loxP site verification
4. Run PCR reaction products on a 2% ethidium bromide agarose gel
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>BioLegend’s Red Blood Cell (RBC) Lysis Buffer (Cat. No. 420301) has been designed,<br />formulated, and tested to ensure optimal lysis of RBCs in single cell suspensions<br />of hematopoietic tissues, such as spleen, as well as RBCs derived from peripheral<br />blood. RBC Lysis Buffer contains ammonium chloride, which has been demonstrated<br />to lyse RBCs with a minimum of harmful effects on lymphocytes</p>

[STEPS]
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>IMPORTANCE: </strong>This review provides a comprehensive comparison of treatment outcomes between robot-assisted laparoscopic surgery (RLS) and conventional laparoscopic surgery (CLS) based on randomly-controlled trials (RCTs).</p>
<p><strong>OBJECTIVES: </strong>We employed RCTs to provide a systematic review that will enable the relevant community to weigh the effectiveness and efficacy of surgical robotics in controversial fields on surgical procedures both overall and on each individual surgical procedure.</p>
<p><strong>EVIDENCE REVIEW: </strong>A search was conducted for RCTs in PubMed, EMBASE, and Cochrane databases from 1981 to 2016. Among a total of 1,517 articles, 27 clinical reports with a mean sample size of 65 patients per report (32.7 patients who underwent RLS and 32.5 who underwent CLS), met the inclusion criteria.</p>
<p><strong>FINDINGS: </strong>CLS shows significant advantages in total operative time, net operative time, total complication rate, and operative cost (<em>p</em> &lt; 0.05 in all cases), whereas the estimated blood loss was less in RLS (<em>p</em> &lt; 0.05). As subgroup analyses, conversion rate on colectomy and length of hospital stay on hysterectomy statistically favors RLS (<em>p</em> &lt; 0.05).</p>
<p><strong>CONCLUSIONS: </strong>Despite higher operative cost, RLS does not result in statistically better treatment outcomes, with the exception of lower estimated blood loss. Operative time and total complication rate are significantly more favorable with CLS. More RCT evidence is needed to be accumulated in order to enable full subgroup analyses on each surgical procedure.</p>
<div class="grammarly-disable-indicator"> </div>

[STEPS]
?. 
?. 
?.
TITLE: iPSC-derived neurons sample preparation for ELISA assays
AUTHORS: Laura Mahoney-Sanchez

[DESCRIPTION]
Cell lysis sample preparation for ELISA assays

[STEPS]
SECTION: Cell lysis
1. Cells were washed with cold PBS then lysed with 300 µL cold RIPA buffer (Thermo Fisher Scientific) supplemented with 1x of a cocktail of phosphatase and protease inhibitors (Life Technologies).

SECTION: Cell lysis
2. Cell lysates were transferred into 1.5ml eppendorf tubes and centrifuged for 5 min at 3500rpm to remove cell debris.

SECTION: Cell lysis
3. Following centrifugation, the supernatant was collected and protein concentration were measured using a BCA or Bradford protein assay (Thermo Fisher Scientific).


SECTION: ELISA assay
4. Equal amounts of protein lysate,  20 µg, were used to perform ELISA assays according to manufacturer's instructions.
TITLE: Th9 Polarization of Mouse CD4+ Cells
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. [Isolation of CD4+ Cells From Lymph Nodes]
Harvest lymph nodes (superficial cervical, mandibular, axillary, inguinal, and mesenteric) from mice.
?. [Isolation of CD4+ Cells From Lymph Nodes]
Tease lymph nodes through a sterile 70-µm nylon cell strainer to obtain single-cell suspensions in complete IMDM containing 10% FCS (complete medium).
?. [Isolation of CD4+ Cells From Lymph Nodes]
Resuspend cells in complete medium and use your favorite method to isolate CD4+cells. Consider using our MojoSort™ Mouse CD4 T Cell Isolation Kit.
?. [Th9 Polarization of CD4+ Cells:]
On day 0, coat 60 x 15mm of plastic petri dishes with anti-mouse CD3ε, clone 145-2C11 (5µg/ml). Incubate at 37°C for 2 hours or 4°C overnight. Aseptically decant antibody solution from the plate. Wash plate 3 times with sterile PBS. Discard liquid.
?. [Th9 Polarization of CD4+ Cells:]
Plate CD4+ cells at 10 x 106/5 ml/dish. Culture cells for 3 days in the presence of anti-mouse CD28, clone 37.51 (5µg/mL), recombinant human TGF-β1 (10ng/mL), recombinant mouse IL-4 (10ng/mL), recombinant mouse IL-2 (20ng/ml), and anti-mouse IFN-γ, clone XMG1.2 (10µg/mL).
?. [Th9 Polarization of CD4+ Cells:]
On day 3, wash cells once and then restimulate in complete medium with 500ng/ml PMA and 500ng/mL ionomycin, in the presence of monensin for 6 hours.
?. [Th9 Polarization of CD4+ Cells:]
After harvesting, the cells are ready for staining.Note: Recombinant human TGF-β is effective for stimulating mouse cells.
TITLE: Modified Frame-projected  Independent Fiber  Photometry (FIP) System_Hardware
AUTHORS: Kenta M. Hagihara

[DESCRIPTION]
This is a step-by-step protocol to build a modified FIP (Frame-projected Independent FP) system. FIP was first implemented and reported by Kim et al. (2016). Their protocol is available here.

We modified previous design so that it can be built mostly with Thorlabs products (still some products from other manufactures are required), which are off-the-shelf and easy to be purchased within a reasonable lead time. The system described in this protocol is designed to record signals from 1)GFP-based sensors, 2)mApple-based sensors,  and 3) iso sbestic signals from up to 4 sites. When applied to measurements of other fluorescent protein-based sensors or spectrally shifted sensors, selection of optical filters and/or excitation light sources should be modified accordingly. Also, the selection
of fiber patch cable should be based on the number of simultaneously recorded locations, and diameter/NA of fiber implants. 

We thank Takeo Katsuki Ph.D., (Thorlabs Japan) and Sho Yagishita M.D., Ph.D., (U.Tokyo, Japan) for sharing their galvano-mirror-based photometry design described in Iino et al. (2020). TK also kindly provided CAD model used in this protocol.

[STEPS]
3. Adding optical filters, collimators, and the objective lens
SECTION: Assemblying optical parts
2. Assemblying the cubes

(This step-by-step part will have corresponding CAD models later.)
4. Assemblysing the fiber positioning module 
5. Assemblysing the CMOS modules
6. Connecting excitation light sources with fibers
SECTION: System Overview
1.       
     

SECTION: Alignment
8. Adjusting angle of collimators so that excitation light coming from fiber patch cables would get roughly uniform.
SECTION: Alignment
7. Adjusting the fiber location so that fiber end makes a crisp image on CMOSs
TITLE: Treg Polarization of Mouse CD4+ Cells
AUTHORS: Sam Li

[STEPS]
?. [Isolation of CD4+ Cells From Lymph Nodes:]
Harvest lymph nodes (superficial cervical, mandibular, axillary, inguinal, and mesenteric) from mice.
?. [Isolation of CD4+ Cells From Lymph Nodes:]
Tease lymph nodes through a sterile 70-µm nylon cell strainer to obtain single-cell suspensions in complete RPMI containing 10% FCS (complete medium).
?. [Isolation of CD4+ Cells From Lymph Nodes:]
Resuspend cells in complete medium and use your favorite method to isolate CD4+cells. Consider using our MojoSort™ Mouse CD4 T Cell Isolation Kit.
?. [Treg Polarization of CD4+ Cells:]
On day 0, coat 12-well plate with anti-mouse CD3ε, clone 145-2C11 (3µg/ml). Incubate at 37°C for 2 hours or 4°C overnight. Aseptically decant antibody solution from the plate. Wash plate 3 times with sterile PBS. Discard liquid.
?. [Treg Polarization of CD4+ Cells:]
Plate CD4+cells at 1.0 x 106/1ml/well. Culture cells for 5 days at 37°C, 5% CO2, in the presence of anti-mouse CD28, clone 37.51 (3µg/mL), recombinant mouse IL-2 (5ng/mL), and recombinant human TGF-β1 (5ng/ml).
?. [Treg Polarization of CD4+ Cells:]
On day 3, if media is yellow, add 2 ml/well of fresh media.
?. [Treg Polarization of CD4+ Cells:]
On day 5, after harvesting, the cells are ready for staining.Note: Recombinant human TGF-β is effective for stimulating mouse cells.
TITLE: Coding Manual for Criminal Justice Reform Policy Documents
AUTHORS: Drew Gitomer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is an adaptation - because we were interested adapting Yanovitzky and Weber's tool for  criminal justice reform.</div><div class = "text-block">All of the policymaking process stuff was kept in, but we modified the list of policy actors, and policy focus, and Appendix C.  </div><div class = "text-block">We also did our coding in NVIvo so removed Appendix D.</div></div>

[STEPS]
TITLE: Protocol 1: Protein-ligand docking
AUTHORS: Phaniendra Alugoju, Vishwambhar Vishnu Bhandare, Vishal S Patil, Krishna Swamy V K D, Prem Kumar Borugadda, Tewin Tencomnao

[DESCRIPTION]
Autodock Vina version 1.1.2 (RRID:SCR_011958) for docking. The grid box's dimensions were fixed at XYZ=30Å × 30Å × 30Å which was found to be the best size for the default exhaustiveness (=8), and the ligand binding site was positioned in the middle of the grid box. The details of spatial dimensions (along XYZ axis) and the grid box’s size which were specified in a configuration file. Using AutoDock vina version 1.1.2's (RRID:SCR_011958) command line interface, docking was accomplished. The log file created listed the binding modes along with their corresponding binding energies. With the help of BIOVIA Discovery studio visualizer 2021, the binding modes were created as a single file in ‘pdbqt’ format, and all non-bonded interactions were recorded.

[STEPS]
TITLE: SARS-CoV-2 Spike Gene N terminal Domain targeted Sequencing 
AUTHORS: Noor Saber Jawad, Nuha Joseph Kandala [Department of Biotechnology

[DESCRIPTION]
We use a simple and effective method for generating 757bp of the N terminal domain of thetheSARS-CoV-2 Spike gene for variant surveillance,      

[STEPS]
SECTION: RNA Extraction
1. The Automated extraction was handled using the ExiPrep™ 96 Lite (A-5250, BIONEER) with the ExiPrep™ Viral DNA/RNA extraction kit (K-4614, BIONEER). 
SECTION: RT-PCR Amplification
2. TaqPath™ COVID‑19 CE‑IVD RT‑PCR Kit (Multiplex real-time RT-PCR test intended for qualitatively detecting nucleic acid from SARS‑CoV‑2) used for viral detection. follow the user manual recommendation as listed in the following link: https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0019215_TaqPathCOVID-19_CE-IVD_RT-PCR%20Kit_IFU.pdf 
SECTION: C-CDNA Synthesis and Quality checking
4. Promega GoScript™ Reverse Transcription Mix with Random Primers system (A2800) is used to generate complementary DNA. following the same kit-recommended procedure. 
SECTION: RT-PCR Amplification
3. Results can be distinguished to: 
1- samples with three positive targets for (ORF1ab, N, and S genes) 
2- sample with two positive targets for (ORF1ab and N genes), negative for theS gene. Failure of Spike gene amplification is referred to as S gene target failure (SGTF) or S gene signal dropdown. 
3- SGTF resulted due to 69/70 codons deletion of Valine and Histidine, respectively.   
SECTION: C-CDNA Synthesis and Quality checking
5. Quality checking is considered for all steps using the Fluorometer Quantus using  Quantifluor dye
SECTION: Primers
6. the forward primer is: SubA_21587F: CCACTAGTCTCTAGTCAGTGTGTT

Reverse primer: SubA_22344R: CCAGCTGTCCAACCTGAAGA

these primers generate an amplicon of 757bp.
SECTION: Primers
6.1. Primer's preparation: 
These primers were supplied by Macrogen Company in a lyophilized form. Lyophilized primers were dissolved in nuclease-free water to give a final concentration of 100pmol/μl as a stock solution. A working solution of these primers was prepared by adding 10μl of primer stock solution (stored at freezer -20 C) to 90μl of nuclease-free water to obtain a working primer solution of 10pmol/μl.
SECTION: Reagent preparation for Amplicon synthesis
7. Amplification reaction carried on using the flowing calculations: 

1- 10 ul of GoTag Green Master Mix, Promega (M7122). 
2- 1 ul of Forward primer 
3- 1ul of Reverse primer 
4- 6 ul of nuclease-free water 
5- 2ul of cDNA template. 
  

SECTION: PCR adopted program and
8. The following program was considered for amplification:
  

SECTION: Gel visualization
9. We use the classic gel visualization method through gel electrophoresis (100-1500 bp ladder gel marker) and gel documentation.  
SECTION: Sequencing
10. We referred our amplicons to a sequencing company (Macrogen, South Korea).