eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<span data-reactid=".0.1.0.1.0.0.0.4.0">This mixture is is used with <a href="https://www.protocols.io/view/Cesium-Chloride-Dialysis-for-Viruses-c7jzkm" target="_blank">Cesium Chloride Dialysis for Viruses</a></span>

[STEPS]
?. 
?. 
?. 
?.
TITLE: UC Davis - Immunohistochemistry Mitochondrial marker
AUTHORS: Jennifer Rutkowsky

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Mitochondria are the power house of the cell. They are distinct organelles with two membranes. Usually they are rod shaped, however they can be round. Several diseases and disorders including schizophrenia, bipolar disorder, dementia, Alzheimer's disease, Parkinson's disease, epilepsy, stroke, cardiovascular disease, retinitis pigmentosa, and diabetes mellitus have all shown association to mitochondrial dysfunction and disorder.</div></div>

[STEPS]
?. Frozen tissue sections (10μM) on slides (tissues embedded in OCT) are then fixed in 4% paraformaldehyde for 5 minutes.
a. Alternatively paraffin embedded sections are deparaffinized through 3 100% xylene 3 min wash
b. Sections were rehydrated through 100%, 95%, 70%, 50%, and 30% then into deionized water (3min each).
c. Antigens are unmasked by steaming in Sodium citrate for 10 min.
?. Wash three times for ten minute in PBS
?. Blocked and permiablize with Block/Perm Solution for 30 minutes.
?. Incubate with anti-mitochondria antibody [MTC02] diluted 1:500 in Block/Perm overnight at 4°C.
?. Wash three times in Block/Perm for 10 minutes
?. Incubated in goat anti-mouse IgG Alexa 555 (1:750) for 1 hour at room temperature.
?. Washing three times in Block/Perm and briefly in PBS
?. Fixed with 4% Paraformaldehyde for 5 minutes.
?. Washed with PBS three times for 10 minutes each wash
?. Coverslip mounted with Prolong gold antifade reagent.
TITLE: Sabouroud’s Dextrose Agar
AUTHORS: Amy Gladfelter

[DESCRIPTION]
This  media  is  used  to  determine  lipid  dependence  of  Malassezia  species.  

[GUIDELINES]
This media is used to determine lipid dependence of Malassezia species.

[STEPS]
1. mycological peptone 10 g

2. glucose 40 g

3. agar 15 g

4. dH2O  1 L

5. Sterilize by autoclaving.
TITLE: Pooled sample testing (VTM/UTM) for SARS-CoV-2 using Magnetic Nanotrap® particles for direct RNA extraction.
AUTHORS: Anurag Patnaik, Ben Lepene, Robert Barclay

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol provides a method for detection of SARS-CoV-2 from pooled Viral transport media and Universal transport media samples using Magnetic Nanotrap</span><span style = "vertical-align:super;">®</span><span> particles.</span></div></div>

[STEPS]
?. [Sample preparation]
Pool 8 clinical VTM / UTM samples. Combine   from each sample into a   or  tube.
500 µl
5 mL
15 mL
?. [Sample preparation]
Add   of Magnetic Nanotrap® particles to the sample.
300 µl
?. [Sample preparation]
Incubate samples with Magnetic Nanotrap®  particles at  for
0 Room temperature
?. [Sample preparation]
Use a magnetic rack to separate the Magnetic Nanotrap® particles from the sample.
?. [Sample preparation]
Discard the supernatant carefully without disturbing the pellet.
?. [Sample preparation]
Add  of 1X PBS to the pellet and resuspend to wash.
500 µl
?. [Sample preparation]
Use a magnetic rack to separate the Magnetic Nanotrap® particles from the sample.
?. [Sample preparation]
Discard the supernatant carefully without disturbing the pellet. If required - use a smaller pipette to remove any residual PBS.
?. [Sample preparation]
Resuspend particle pellet in   of extraction buffer (Quick vortex if required).
50 µl
?. [Sample preparation]
Heat samples at   for
95 °C
This step can be performed on a heat block or thermocycler.
?. [Sample preparation]
Transfer the resuspension mix to a  OR    microcentrifuge tube.
0.5 mL
1.5 mL
Do not heat samples in a    or larger tube. The heat distribution required for lysis is not adequate in larger tube sizes.
?. [Sample preparation]
Use a magnetic rack to separate the Magnetic Nanotrap® particles from the sample.
?. [Sample preparation]
Collect the supernatant. The sample is ready for analysis.
?. [RT-PCR detection]
Use any SARS-CoV-2 RT-PCR detection kit. Follow manufacturer instructions to set up the RT- PCR
TITLE: Converting ssDNA oligos to dsDNA with T4 DNA polymerase
AUTHORS: Alex N Nguyen Ba

[DESCRIPTION]
This protocol allows one to convert ssDNA to dsDNA oligos. In principle, one can buy two complementary oligos and anneal them. However, there are a few cases where randomized bases are desired, and therefore complementary oligos can not be ordered. The protocol works similarly to PCR, except that the reaction is not performed at the high temperature of typical thermocycling, because the oligos are short and their melting temperature can be in the order of the extension temperature of high-fidelity polymerases. This protocol is therefore particularly useful when creating barcodes or when short dsDNA fragments containing degenerate bases are desired.

[STEPS]
SECTION: Primer design
1. Design your target single-stranded DNA oligo such that the 3' end contains the reverse complement of an extending oligonucleotide. For example, appending "GTCATAGCTGTTTCCTG" to the end of your oligo will allow an oligonucleotide matching the M13 Reverse (-27) primer to extend it (5'-CAGGAAACAGCTATGAC-3').
SECTION: Oligo annealing
4. Mix in a PCR tube 5 µL of a 10 micromolar (µM) stock of the target oligo with 10 µL of a 10 micromolar (µM) stock of the extending oligo. Add 27ul of molecular biology grade water.

SECTION: Oligo annealing
5. Add 5 µL of  . Mix well by vortexing.

SECTION: Oligo annealing
6. Place the tube in a thermocycler and run the following protocol:
1. 95 degrees for 10 seconds
2. Decrease by 1 degrees
3. Repeat step 2 every 10 seconds, for 90 cycles.
4. Hold at 4 degrees.

SECTION: Primer design
2. Resuspend your oligonucleotides in 1/10th TE (10mM Tris, 0.1mM EDTA pH 8) to a final concentration of 100 micromolar (µM) to form your stock oligonucleotide solutions.

SECTION: Primer design
3. Make working oligonucleotide solutions by diluting the stock oligonucleotides to a final concentration of 10 micromolar (µM) in water.

SECTION: Oligo extension
7. Add 2.5 µL   (10 millimolar (mM) stock) and mix well by vortexing.
SECTION: Oligo extension
8. Go to the thermocycler and prepare the reaction cycle. When ready, start the protocol and pause when the block reaches 0 degrees (when Step 1 begins).

1. 5 minutes at 0 degrees C.
2. 5 minutes at 22 degrees C.
3. 30 minutes at 37 degrees C.
4. Hold at 0 degrees.
SECTION: Oligo extension
9. Add 0.5 µL of   and mix well.

SECTION: Oligo extension
10. Place the tube in the thermocycler and unpause the run.
SECTION: Oligonucleotide cleanup
11. As soon as the reaction is completed, add   to 10 millimolar (mM).

SECTION: Oligonucleotide cleanup
12. Use a PCR purification kit to purify the oligo. Ensure the kit is able to purify short oligonucleotides if needed (a kit such as   can be suitable).
TITLE: Leica SP8 Confocal Imaging
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol describes the basic setup and scanning using the Leica SP8 confocal microscope of fluorescently-labeled mouse brain tissue sections mounted on positively charged 1” x 3” microscope slides.

Note: 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.

[STEPS]
TITLE: In vivo Ephys: Spike Sorting Protocol
AUTHORS: Sasha Burwell

[DESCRIPTION]
This protocol details the spike sorting procedures used on the collected electrode recording data.

[STEPS]
SECTION: Spike Sorting Protocol
1. For more information on setting up and using Spyking Circus, see: https://spyking-circus.readthedocs.io/en/latest/code/index.html .
SECTION: Spike Sorting Protocol
1.1. Make a copy of the continuous.dat data file from your recording into a new “sorted_data” folder.

 

SECTION: Spike Sorting Protocol
2. Add a .params file with the same name as the .dat file to the folder (i.e., continuous.params). The continuous.params used for these experiments:

 

SECTION: Spike Sorting Protocol
3. The .params file needs to have correct path to a .prb file (the mapping of the electrode you are using). We recorded with bundles of 16 individual electrodes, and used the following .prb file:

 


SECTION: Spike Sorting Protocol
4. Launch the Spyking Circus GUI from the anaconda terminal.
SECTION: Spike Sorting Protocol
5. Run the sorting in the Main Algorithm tab.
SECTION: Spike Sorting Protocol
6. When the next window pops up, click “Suggest pairs”, “Select pairs”, then “Merge and finalize”.
SECTION: Spike Sorting Protocol
5.1. Browse → select continuous.dat file.
SECTION: Spike Sorting Protocol
5.2. Increase CPUs as desired, then click “Run”.
SECTION: Spike Sorting Protocol
7. To view results: View Results tab → Browse → select the analyzed continuous.dat file.
SECTION: Spike Sorting Protocol
7.1. Options:

Matlab GUI
Extension = “merged”
SECTION: Spike Sorting Protocol
7.2. Click “Run”.
SECTION: Spike Sorting Protocol
7.3. See https://spyking-circus.readthedocs.io/en/latest/GUI/sorting.html for detailed documentation on what each panel of the GUI does.
SECTION: Spike Sorting Protocol
8. Sorting:
SECTION: Spike Sorting Protocol
8.1. Set bin size to 1, click “Big ISI”, and update the scale so you can see whole waveforms (zooming out usually works).
SECTION: Spike Sorting Protocol
8.2. Go through all templates.
SECTION: Spike Sorting Protocol
8.3. First pass: Kill a cell if it has above a 1% incidence of refractory period violations.

 

SECTION: Spike Sorting Protocol
8.4. Second pass: merge templates that you think are the same cell, using the two template view.

Criteria used: similar waveforms, cross correlation dropping to zero at zero, coefficient of similarity close to 1.
Sometimes you have to merge more than two templates for one cell.
Merge template with fewer spikes into the one with more spikes.
SECTION: Spike Sorting Protocol
8.5. Third pass: kill units that don’t meet requirements (too sparse, not spiking consistently across the whole session, auto correlation doesn’t go to zero, RPV has gone above 1%).
SECTION: Spike Sorting Protocol
9. Add suffix (ex, ‘v1’ for first attempt at sorting) and save.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Protocol for Dephosphorylation of 5&acute;-ends of DNA using CIP in Restriction Enzyme Reaction. Uses the Calf Intestinal&nbsp;Alkaline Phosphatase &nbsp;(CIP - M0290)

[GUIDELINES]
<p><strong>Dephosphorylation of 5' -ends of DNA in Restriction Enzyme Reaction</strong></p>
<ul>
<li>The phosphate can be added directly into the digestion reaction during or after DNA digestion</li>
<li>CIP is active in all NEB restriction enzyme buffers</li>
<li>DNA purification is required before ligation</li>
</ul>

[STEPS]
?. 
?. 
?.
TITLE: QIAamp DNA Extraction Protocol
AUTHORS: Vicky Ooi, Lee McMichael, Margaret E. Hunter, Aristide Takoukam Kamla, Janet M. Lanyon

[DESCRIPTION]
Dugong faecal DNA was extracted using the QIAamp Fast DNA Stool Mini Kit (#51604, Qiagen, Germany). The "Protocol: Isolation of DNA from Stool for Human DNA Analysis" was optimised for DNA extraction from dugong faeces.

[STEPS]
1. Use a sterile blade to scrape off 220 mg of faecal material from the outside surface of a stool and then transfer it into a 2 mL microcentrifuge tube.
2. Transfer the faecal material into a mortar and grind the faeces into powder with liquid nitrogen.
3. Add 500 μL of InhibitEX buffer to the mortar containing the faecal material to further grind and mix in the buffer with the ground faeces. Transfer the liquid back into the 2 mL tube.
4. Add another 500 μL of InhibitEX buffer to the mortar to mix in any leftover faecal material on the mortar. Transfer the liquid back into the 2 mL tube.
5. Vortex continuously for 1 min or until the solid material is thoroughly homogenised.
6. Centrifuge the sample at 20,000 g (~14,000 rpm) for 2 min to pellet stool particle.
7. Pipette 25 μL of Proteinase K into a new 2 mL tube.
8. Pipette 800 μL of supernatant from the centrifuged homogenate into the 2 mL microcentrifuge tube containing Proteinase K.
9. Add 800 μL of Buffer AL and vortex for 15 s.
10. Incubate at 70°C for 10 min. Then, centrifuge briefly to remove drops from the inside of the tube lid.
11. Split the lysate into two 2 mL tubes (~813 μL each tube).
12. Add 400 µL of 95% ethanol to both tubes containing the lysate (thus, 800 µL of ethanol overall) and mix by vortexing.
13. Carefully apply 600 μL of lysate to the QIAamp spin column. Close the cap and centrifuge at 20,000 g (~14,000 rpm) for 1 min. Place the QIAamp spin column in a new 2 mL collection tube and discard the tube containing the filtrate.
14. Repeat step 13 until all the lysate has been loaded on the column.
15. Carefully open the QIAamp spin column and add 500 µL of Buffer AW1. Centrifuge at 20,000 g (~14,000 rpm) for 1 min. Place the QIAamp spin column in a new 2 mL collection tube and discard the collection tube containing the filtrate.
16. Carefully open the QIAamp spin column and add 500 µL of Buffer AW2. Centrifuge at 20,000 g (~14,000 rpm) for 3 min. Place the QIAamp spin column in a new 2 mL collection tube and discard the collection tube containing the filtrate.
17. Centrifuge at 20,000 g (~14,000 rpm) for 3 min to eliminate the chance of possible Buffer AW2 carryover.
18. Transfer the QIAamp spin column into a new, labelled 1.5 mL microcentrifuge tube and pipette 100 µL of Buffer ATE directly onto the QIAamp membrane to elute the DNA from the spin column into the 1.5 mL Eppendorf LoBind microcentrifuge tube. Incubate for 1 min at room temperature, then centrifuge at 20,000 g (~14,000 rpm) for 1 min to elute DNA.
19. Store the DNA isolate at -20°C for use within a week or at -80°C for longer-time storage.
TITLE: Effectiveness of exercise therapy and self-management education to improve physical activity levels in patients with acute exacerbations of chronic  obstructive pulmonary disease: protocol of a systematic review and meta-analysis
AUTHORS: Koki  Yamamoto, Mami Takayama, Tadayoshi Nonoyama, Yusuke Kon, Yoshiki  Saimon, Takashi  Kitagawa

[DESCRIPTION]
We aimed to evaluate the effectiveness of exercise therapy and self-management education in improving physical activity levels (PA) in patients with acute exacerbations of chronic obstructive pulmonary disease (AECOPD) and to provide future research directions. We plan to include all randomized controlled trials (RCTs) of exercise therapy and self-management education or either intervention alone designed to improve PA in patients with AECOPD. We will determine the differential effects of exercise therapy and self-management education interventions on daily steps, moderate to vigorous activity time (MVPA), and sedentary equivalent time (SB) (P: AECOPD, I: 1. exercise therapy, 2. self-management education, 3. exercise therapy and self-management education, C: usual care, O C: usual care, O: steps per day, MVPA, SB). In this review, we plan to use five databases. Two reviewers will screen articles to identify studies for inclusion.For risk of bias assessment, we will use Risk of Bias 2 tool.

[STEPS]
TITLE: Dissolved Fe(II/III) colorimetric assay using a plate reader (96-well plate)
AUTHORS: Jian Gong

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes the adaption of an Iron assay originally described in </span><a href="https://doi.org/10.1016/S0883-2927(99)00097-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Viollier 1999</span></a><span> and </span><a href="https://doi.org/10.1021/ac60289a016" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Stookey 1970</span></a><span>, modified for use on a multi-mode plate reader spectrophotometer (BioTek, Synergy 2, Winooski, VT, USA), using standard </span><a href="https://www.sigmaaldrich.com/catalog/product/SIGMA/CLS9017?lang=en&region=US" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">96-well plates</span></a><span> for rapid measurements of 1 mL water samples. This assay measures the concentration of both Fe(II) and Fe(III) ions in solution.</span></div><div class = "text-block"><span>Samples for this assay should be filtered (</span><a href="https://www.sigmaaldrich.com/catalog/product/aldrich/wha67802502?lang=en&region=US" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">0.2 µm syringe filter</span></a><span>), measured or diluted to 1.20 mL volume and placed in </span><a href="https://www.sigmaaldrich.com/catalog/product/sigma/t9661?lang=en&region=US" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">1.5 mL microcentrifuge tubes</span></a><span>. Care should be taken to avoid oxidation of Fe(II) ions by oxygen in the atmosphere, either by performing the steps quickly under a stream of N</span><span style = "vertical-align:sub;">2</span><span> gas right after sampling or by performing the first step of this assay inside an anaerobic chamber.</span></div></div>

[STEPS]
?. Measure absorbance A1: A1 is measured immediately after adding  of reagent A to  of filtered/filtered-diluted samples or undiluted standard solutions. Mix the mixture well with the pipette by gently pipetting up and down a few times. Load three  mixtures onto a 96-well plate to make triplicate measurements on the spectrophotometer.
120 µl
1.2 ml
200 µl
?. Reduction and measure absorbance A2: collect  of the remaining mixture after step 1, then add  of reagent B. Mix with the pipette and begin the timer. The solution is allowed to react forto complete the reduction from Fe(III) to Fe(II). At the 10 min mark, add  reagent C, mix with the pipette and immediately measure A2 by loading triplicate  mixtures on the well plate.
640 µl
120 µl
40 µl
200 µl
?. Steps 2 is repeated once only for the standards, from which A3 is measured and α calculated. In the procedure above α should be close to 0.8. The goal of executing this step is to obtain a realistic α together with estimates of its uncertainty during the assay procedure.
TITLE: Recombining DNA by Gibson Assembly
AUTHORS: Tobias von der Haar

[DESCRIPTION]
Our protocol for producing recombinant DNA via Gibson Assembly, based on a home-made Gibson Master Mix.

[STEPS]
1.  
 
 
SECTION: Preparation of 5x Isothermal Buffer
2. Mix the following to give 1 ml of 5x Isothermal Buffer:
450 μl 50% PEG 8000
250 μl 2M Tris-HCl pH 7.5
100 μl 500 mM MgCl2
50 μl 1M DTT
100 μl 50 mM NAD
10 μl each of 100 mM ATP, CTP, GTP and TTP (PCR grade)
10 μl sterile water
 

SECTION: Preparation of Gibson Master Mix
3. Mix the following reagents:
160 μl of 5x Isothermal Buffer
3.2 μl of 1U/μl T5 exonuclease (diluted 1:10 in water from the 10 U/μl stock)
10 μl of 2U/μl Phusion polymerase
80 μl of 40 U/μl Taq ligase
346.8 μl deionised water
Freeze in 15 μl aliquots (one 15 μl aliquot is ready-to-use for one Gibson assembly reaction)
 
 
 
SECTION: Gibson Assembly
4. Preparation: You need to have prepared two or more fragments of DNA which you wish to recombine, and which have 25-40 nucleotide homologous regions at their ends. For example, this could be a linearised vector and a PCR product which has sequences corresponding tot he ends of the linearised vector introduced via the primers.
 
Mix the fragments to be assembled in equimolar ratios in a total volume of 5 μl. Add this mixture to one 15 μl aliquot of Gibson Master Mix.
Incubate the reaction in a 50°C water bath or in a PCR machine at 50°C  for one hour.
Use 2-10 μl of the assembly reaction for a bacterial transformation.
TITLE: Detection of marbled crayfish Procambarus fallax
AUTHORS: Alexander Eiler, Eivind Stensrud, Omneya Osman

[DESCRIPTION]

Taqman QPCR assay for marbled crayfish Procambarus fallax

 




[GUIDELINES]
Handling high concentration of positive controls was performed in a post-PCR room which is physically separated from the pre-PCR room to avoid contamination. 
Always add your samples first and seal them before adding the serial dilutions of positive control (standard) at the end.

[STEPS]
SECTION: DNA extraction
1. 
A tissue of marbled crayfish was extracted with DNeasy blood and tissue extraction kit
https://www.qiagen.com/us/shop/pcr/dneasy-blood-and-tissue-kit/
The quality of DNA was checked by nanodrop.

SECTION: DNA extraction
3. Primers

 

SECTION: DNA extraction
4. 2 µL Standard dilution

DNA of marbled crayfish was serially diluted from 1e2-1e-4 for qPCR experiment. 
SECTION: DNA extraction
5. PCR mixture
 

SECTION: DNA extraction
6. - °C Amplification conditions

 

SECTION: DNA extraction
7. qPCR was performed in BioRad qPCR machine CFX96.

 


SECTION: DNA extraction
8. 
 

SECTION: DNA extraction
9. Primer validation
DNA of related crayfish species were tested to ensure primers specificity.
 
 

SECTION: DNA extraction
2. Internal control: http://www.primerdesign.co.uk/assets/files/internal_control_handbook_dna.pdf?timestamp=1469446474
TITLE: SOP for LBP ELISA after DSS-induced injury
AUTHORS: Malu G Tansey

[DESCRIPTION]
SOP for LBP ELISA after DSS-induced injury

[STEPS]
SECTION: Protocol
1. Concentrations of plasma LPS-binding protein (LBP) were examined via Mouse LBP SimpleStep ELISA according to the manufacturer’s protocol (Abcam, #ab269542).
SECTION: Protocol
2. Plasma samples were prepared at a 1:16,000 dilution.
SECTION: Protocol
3. Standards, antibody cocktail containing detector and capture antibodies, and 1x wash buffer were prepared according to the manufacturer’s protocol.
SECTION: Protocol
4. In the plate, 50µL of standard or sample was added to appropriate wells in duplicate.
SECTION: Protocol
5. 50µL of antibody cocktail was added to all wells.
SECTION: Protocol
6. The plate was then incubated at RT for 1 hour on a plate shaker at 400 rpm.
SECTION: Protocol
7. Wells were decanted and washed three times with 150µL 1x wash buffer.
SECTION: Protocol
8. 100µL of TMB Development Solution was added to each well and allowed to incubate in the dark for 12 minutes with shaking at 400rpm
SECTION: Protocol
9. 100µL of Stop Solution was added to each well and placed on the shaker for 1 minute at 400 rpm to mix.
SECTION: Protocol
10. Absorbance of 450nm light was recorded with a FLUOstar Omega microplate reader (BMG Labtech).
SECTION: Protocol
11. The standard curve was created by plotting the blank-subtracted average LBP standard absorbance against the standard LBP concentrations.
SECTION: Protocol
12. Sample LBP concentrations were calculated by interpolating the blank-subtracted average absorbance values against the standard curve, multiplied by the dilution factor.
TITLE: 反转录cDNA
AUTHORS: 张 雪

[STEPS]
?. [方法A.Ominiscript kit]
AB110×buffer RT2µl2dNTP mix(5mM) 2µl3oligo-dT primer(10µM)2µl4RNase inhibitor1µl5Ominiscript reverse transcriptase1µl6Template RNA2µg7RNase-free H2Oadd to 20µl
RNase-free枪头
AB110×buffer RT2µl2dNTP mix(5mM) 2µl3oligo-dT primer(10µM)2µl4RNase inhibitor1µl5Ominiscript reverse transcriptase1µl6Template RNA2µg7RNase-free H2Oadd to 20µl
?. [方法A.Ominiscript kit]
混匀，37°C，1h
37 °C
?. [方法A.Ominiscript kit]
测浓度
?. [方法B. Thermo kit合成cDNA]
使用8联管便于后续用PCR仪控温  AB1total RNA0.1ng～5µg2Oligo(dT)18  Primer1µl3RNase-free H2Oadd to 12µl
RNase-free枪头
AB1total RNA0.1ng～5µg2Oligo(dT)18  Primer1µl3RNase-free H2Oadd to 12µl
?. [方法B. Thermo kit合成cDNA]
混匀，65°C，5min，此后在冰上操作
65 °C
?. [方法B. Thermo kit合成cDNA]
向上述体系继续加入以下组分  AB15×reaction buffer4µl2RNase inhibitor1µl310mM dNTP mix2µl4reverse transcriptase1µl
on ice
AB15×reaction buffer4µl2RNase inhibitor1µl310mM dNTP mix2µl4reverse transcriptase1µl
?. [方法B. Thermo kit合成cDNA]
混匀，45°C，1h
45 °C
?. [方法B. Thermo kit合成cDNA]
72°C，5min
72 °C
?. [方法B. Thermo kit合成cDNA]
测浓度