eamag/protocols-with-steps huggingface dataset:
TITLE: Method of inventory and monitoring of bat fauna in forests by using mist nets
AUTHORS: Alona Prylutska, Anton Vlaschenko

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The most common approaches for summer bat monitoring in Europe are acoustic recording and captures from roosts. However, these methods provide only fragmentary data about species richness and abundance, without information about population structure. In order to obtain complex information about sex and age structure, in 2008 we developed method wich implies mist netting for a primary inventory and following monitoring of bat fauna in forests.  </div><div class = "text-block">In short period of time (20 days) we revealed all bat species known for the territory  of the National Park “Homilsha forests” (Kharkiv region, Ukraine).</div><div class = "text-block">The method described here was sucesfully used by our team in inventory bat fauna in Kharkiv region, Ivano-Frankivsk region, Chornobyl Exclusion Zone (Ukraine), Nature reserve "Brynsky forest", National park "Smolensk lakeland", Oksky Nature reserve, Voronezh Nature Resrve (Russia). </div><div class = "text-block">Compare to bat detectors mist nets are cheaper technique for bat surveys, but need more time input.</div></div>

[STEPS]
?. Choose the period for bats captures. We consider July as the most effective period for mist-netting when  young individuals start to fly, but the autumn migration has not started yet.
?. Select  study plot, about 400-500 ha with different habitats (e.g. lake,river shores, forest edges and closed forest roads). Choose 9 capture points (the distance between capture sites should be no less than 600 and could be up to 3000 m).
?. Use  nylon mist nets (12 x 3 m, mesh size 15 mm). Install the net from sunset till 10–30 min beforesunrise.  Stay near to the net during all night, bacause bats can escape from the net.  Keep bats in textile bags near the net throughout the night.
?. Conduct two rounds of mist-netting at each capture site (two nights at every point). First 9 capture nights at 9 points, and then second round of the same 9 points. It will be 18 captures totally.
?. Record species, sex, age and identified breeding status of adult individuals of both sexes. All animals after biometrical process release as soon as possible (at dark time, of cause).
TITLE: Protocol 1
AUTHORS: lukasz.lukjaniuk 

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

[STEPS]
?. [Ekstrakcja]
Add1 in Store in
1 µl
25 Room temperature
?. [Ekstrakcja]
After step 1 rotate
10 33
?. [Ekstrakcja]
AV_2^N
[Buffor]
?. [Ekstrakcja]
?. 
?.
TITLE: 16S PCR
AUTHORS: Stephanie  Clouser

[DESCRIPTION]
A protocol for 16S PCR as outlined by the HUCK Genomics Core.

[STEPS]
SECTION: Before You Begin
1. Prepare your workspace
1.     Turn on PCR Clean Hood blower and white light.
2.     Clean the cabinet with 70% ethanol, including the work surface, walls, and glass.
3.     Turn the dial and run the UV light for 15 minutes.                                     
 

4.     Once the timer is up and the UV light turns off, you are ready to begin.
5.     Clean all materials with 70% ethanol before putting them into the cabinet.
SECTION: Before You Begin
2. Gather materials 
Place your materials on the bench next to the PCR Clean hood. Clean lab bench well with 70% ethanol before use.
a.     Invitrogen Platinum SuperFi Master Mix
b.     515f Parrada Primer at 10uM
c.     806R Apprill Primer at 10uM
d.     Sterile PCR H2O
e.     PCR Plate/Tube Rack
f.      Adhesive Film
g.     96-well PCR Plate or 8-well PCR tube (depending on number of samples)
h.     Pipette Tips (10ul, 20ul, 100ul, 1000ul)
i.      Micropipettes (P10, P20, P100, P1000)
j.      VWR marker
k.     Sterile Centrifuge Tubes
SECTION: Master Mix Preparation
3. 1.     In a sterile centrifuge tube labeled “Master Mix/MM” combine: 

10 µL /sample Invitrogen Platinum SuperFi Master Mix, 
0.4 µL /sample 515F Parrada Primer, 
0.4 µL /sample 806R Apprill Primer, and 
7.2 µL /sample Sterile PCR H2O. 

        Prepare enough master mix for 100 samples if running a full plate:
2.     Vortex and spin down master mix when complete.12500 rpm, 2 min, 25 °C 



 

SECTION: PCR Reaction
4. 1.     Add 18 uL of Master Mix to each PCR tube.
2.     Add 2 uL of the extracted DNA to each respective PCR tube. Be sure to follow the plate map, create a new PCR plate map, and/or label tubes.
3.     Run Thermocycler Program:
4.     When cycles are complete, centrifuge tubes. Tubes can then be stored at 4°C until ready to run gel or AMPure.
TITLE: Fungal DNA extraction for  Nanopore sequencing
AUTHORS: Eri  Ogiso-Tanaka, Hiyori   Itagaki, Muneyuki   Ohmae, Tsuyoshi   hosoya, Kentaro   Hosaka

[DESCRIPTION]
This protocol is intended for extraction of high molecular weight DNA from fungal samples.

[BEFORE_START]
Cut the tip of the pipette man tips in advance to make the outlet wider.

[STEPS]
1. The frozen mycelium samples are ground to fine powders using a mortar and pestle in liquid nitrogen to avoid raising the temperature of the samples for high molecular weight DNA extraction. 

2. Add 20 mL of pre-warmed (60 °C) lysis buffer with 800 µL ProteinaseK (FUJIFILM Wako Pure Chemical Co., Ltd. Japan) and 50 µL RNase A (Nippon Gene Material Co., Ltd. Japan) in a glass beaker. 

20 mL lysis buffer [2% CTAB, 100mM TrisHCl, 20mM EDTA, 1.4M NaCl, 1% PVP)]  
800 µL ProteinaseK
50 µL RNase A 
60 °C 

3. The mixture was incubated at 55 °C in a water bath with shaking for 5 min.
 55 °C
 5 min.
4. Add  1 vol phenol/chloroform/isoamylic alcohol (PCI) 25:24:1 and mix by inversion.  


8. Rinse pellet with 1mL 75% ethanol at RT. 
1 mL  75% ethanol

11. Air dry DNA pellet.

 

5. Extracted DNA was quantified spectrophotometrically with NanoDrop (Thermo Fisher Scientific, USA) and in a Qubit 2.0 fluorometer (Thermo Fisher Scientific). 
6. The quality of DNA was estimated using Genomic DNA ScreenTape assay with 2200 TapeStation system (Agilent Technologies, Germany).
7. 
4.1. Centrifuge 12000 rpm (13000 g) with soft/slow break function for  20 min at RT.

 12000 rpm 13000 x g 
20 min 
Room temperature 

4.1. 
4.2. Remove aqueous layer to new 50 mL tube.
4.3. Repeat step 4
5. Add 1 vol chloroform and mix by inversion.

 

5.1. Centrifuge 14000 rpm (17800 g) with soft/slow break function for  10 min at RT.
14000 rpm 17800 x g 
10 min
Room temperature 

5.2. Remove aqueous layer to new 50 mL tube.
5.3. Repeat step 5
6. Add 1 vol isopropanol and mix gently by inversion.
7. Centrifuge 14000 rpm for  5 min at 4 °C. Remove and discard the supernatant.
14000 rpm 17800 x g 
5 min
4 °C 

9.  All aqueous and pellet to new 2 mL tube.
10. Spin down and remove supernatant.
12. Resuspend DNA in sterile 50uL 10mM Tris-HCl for 1 hr~ at RT in dark.
50 µL Tris-HCl
Room temperature 
60 min~60 min 

 

15. Store at 4 °C until library preparation. (at -20 °C for long-term storage)

13. Check the quality and concentration of DNA using spectrophotometrically with NanoDrop (Thermo Fisher Scientific, USA) and in a Qubit 2.0 fluorometer (Thermo Fisher Scientific).
14. Check the DNA degradation using Genomic DNA ScreenTape assay with 2200 TapeStation system (Agilent Technologies, Germany).
TITLE: SNA (Synthetic Nutrient Deficient) Agar for Identification fungi
AUTHORS: Gabriela Paredes

[DESCRIPTION]
SNA (Spezieller Nährstoffarmer Agar), or Synthetic Nutrient Deficient Agar, is a specialized culture medium designed for the identification and study of fungi such as Fusarium and Cylindrocarpon. This medium is nutrient-deficient, promoting specific morphological characteristics in these fungi that are critical for accurate identification. SNA is frequently used in mycological research and diagnostics, particularly in laboratories focused on plant pathology and fungal taxonomy. If the goal is to observe and document specific growth patterns, sporulation, or other morphological traits of these fungi under nutrient-limited conditions, SNA agar provides an optimal environment.

When Not to Use SNA Agar:
Isolation of Nutrient-Demanding Fungi:
For fungi that require richer nutrient environments for growth, such as certain saprophytic or pathogenic fungi not belonging to Fusarium or Cylindrocarpon, SNA agar would not provide the necessary nutrients for their development.

Rapid Growth and Mass Production:
If the objective is to rapidly grow and mass-produce fungal cultures for spore collection or other applications, a nutrient-rich medium such as Potato Dextrose Agar (PDA) or Sabouraud Dextrose Agar (SDA) would be more appropriate

[BEFORE_START]
Before starting the preparation of SNA medium, ensure that all necessary materials and equipment are available and properly sterilized. Measure out all the required chemical components accurately.

[GUIDELINES]
Aseptic Technique: Ensure that all steps, especially post-autoclaving, are conducted under aseptic conditions to prevent contamination.
Homogeneous Mixing: Stir the medium well after dissolving the components and before autoclaving to ensure uniform distribution.
Temperature Control: Allow the medium to cool to approximately 50°C before pouring to prevent the formation of condensation in Petri dishes and to maintain the sterility of the medium.

[STEPS]
SECTION: Dissolve SNA Components:
1. In a 1-liter Erlenmeyer flask, dissolve the following components in 1 liter of distilled water (dH2O):
1 g  of KH2PO4 (Potassium dihydrogen phosphate)
1 g  of KNO3 (Potassium nitrate)
0.5 gof MgSO4·7H2O (Magnesium sulfate heptahydrate)
0.5 gof KCl (Potassium chloride)
0.2 gof Glucose
 0.2 µLof Sucrose
20 g of Agar






SECTION: Sterilize:
2. Autoclave the solution at 121°C for 20 minutes to sterilize the medium.
After autoclaving, allow the medium to cool to around 50°C before pouring it into Petri dishes.
SECTION: Pour into Petri Dishes:
3. Work aseptically to pour the medium into sterile Petri dishes.
Allow the agar to solidify at room temperature before use.
TITLE: Adult Mouse Spleen Dissociation (On ice)
AUTHORS: Andrew Potter

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol used to dissociate adult (8-10 wk) mouse spleen into single cells. Attained >95% viability, a variety of cell sizes, and ~10 million cells from 12 mg tissue.</div></div>

[STEPS]
?. [Isolate Tissue]
Using sterile forceps, transfer spleen to petri dish on ice. Remove excess DPBS using pipet. Chop whole spleen coarsely for 45 sec using razor blade on petri dish on ice until a fine paste. Manipulate tissue with forceps while mincing with razorblade to thoroughly break up.
[mince on ice]
?. [First layer of dissociation]
Weigh out 12 mg of minced spleen on petri dish. Using razor blade, transfer to 1.5 mL tube containing 1 mL of enzyme mix on ice.
?. [First layer of dissociation]
Incubate tube on ice for 10 minutes. Triturate 10X every 2 mins and shake every min.
[Incubate on ice]
[triturate 10X every 2 min]
[Shake every min]
?. [First layer of dissociation]
After 10 mins of digestion, let tissue chunks settle for 1 min on ice & save 80% of supernatant with released cells & apply cells to 30 µM filter on 50 mL conical, on ice. Rinse filter with 5 mL ice-cold PBS/0.04% BSA. Leave filter and 50 mL conical on ice, it will be used for the steps as well.
[ice-cold PBS/BSA 0.04%]
?. [Second layer of dissociation]
Add additional 1 mL enzyme mix to tissue chunks.
?. [Second layer of dissociation]
Continue to triturate 10x every 2 minutes and shake every minute while incubating on ice, for 10 additional minutes (21 min total time).
[Incubate on ice]
[triturate 10x every 2 min]
[shake every min]
?. [Second layer of dissociation]
After 21 min total time, triturate 10x and add entire volume of remaining digest mix to the same 30 µM filter on 50 mL conical as in the previous step. Rinse filter w/5 mL ice-cold PBS/0.04% BSA.
[ice-cold PBS/BSA 0.04%]
?. [RBC Lysis]
Transfer flow-through to 15 mL conical. Spin 650 g for five minutes at 4 °C. After spin, remove supernatant, leaving 100 µL volume in 15 mL conical tube.
[spin 650 g for 5 min]
?. [RBC Lysis]
Perform RBC lysis: add 1 mL RBC lysis buffer to the 100 µL of cells and triturate 10X. Let sit 3 minutes on ice. After incubating 3 min in RBC lysis buffer, add 13 mL ice-cold PBS/BSA 0.04% in the 15 mL conical to dilute the RBC lysis buffer. Pipet mix.
[incubate on ice]
[ice-cold PBS/BSA 0.04%]
[RBC lysis buffer]
?. [Preparing cells for downstream analysis]
Spin 650 g for 5 mins at 4 °C. Remove supernatant and re-suspend in 1 mL ice-cold PBS/BSA 0.04%.
[spin at 650 g for 5 min]
?. [Preparing cells for downstream analysis]
Examine cells using hemocytometer with trypan blue. Adjust concentration to 1000 cells / µL for 10x chromium or 100 cells / µL for DropSeq.
?. [Isolate Tissue]
Isolate whole spleen and immerse/transport in ice-cold DPBS.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>    Potentiometric titrationis can be used to determine the effective content of general chemical . The method offers many advantages, including simplicity, speed, easier end-point determination and more accurate results.</p>

[GUIDELINES]
<p>Composite platinum (Pt) electrodes or a monomer platinum (Pt) indicator electrode and reference electrode were used to measure available chlorine, available iodine and hydrogen peroxide content.A composite water phase pH electrode(containing a pH indicator electrode and a Ag/AgCl reference electrode)or an aqueous phase pH-indicating electrode and reference electrode monomer were used to measure glutaraldehyde content.A composite non-aqueous phase pH electrode (containing a pH indicator electrodeand a Ag/AgCl reference electrode)was used to measure the chlorhexidine content. Finally, a surfactant aqueous phase titration indicator electrode and a reference electrode were used to measure  benzalkonium bromidecontent.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Mouse Brain Perfusion and Flash Freezing 
AUTHORS: Allen Institute

[DESCRIPTION]
This protocol is used for transcardial perfusion with HEPES-Sucrose Cutting Solution (Referred as HEPES hereafter), followed by fast brain dissection and flash freezing.

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: Protocol for Exo-CIP™ Rapid PCR Cleanup (#E1050)
AUTHORS: New England  Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Exo-CIP™ Rapid PCR Cleanup Kit</span><span></span></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Rapidly degrade residual PCR primers and dephosphorylate excess dNTPs after amplification</li><li style = "counter-reset:ol0;list-style-type:disc;">Reaction complete in 4 minutes</li><li style = "counter-reset:ol0;list-style-type:disc;">Thermolabile formulation can be heat inactivated in 1 minute at 80°C</li><li style = "counter-reset:ol0;list-style-type:disc;">PCR product can be used directly in downstream applications</li><li style = "counter-reset:ol0;list-style-type:disc;">Compatible with commonly-used reaction buffers</li></ul></div></div>

[STEPS]
?. Transfer  of PCR product to a new PCR tube and add  of Exo-CIP A and Exo-CIP B respectively. The final volume is .
5 µl
1 µl
7 µl
?. Mix thoroughly and briefly centrifuge at .
Centrifuge: 1000 34
?. Incubate the reaction tube for   at  followed by  at .
37 °C
80 °C
?. Submit   or less (in a range of 15-200 fmol)* of treated PCR product for sequencing using  BigDye™ Terminator v3.1 Cycle Sequencing Kit or store the treated samples at   for longer term storage.
3 µl
-20 °C
* A simple way to determine the amount of your amplicon is to load    on an agarose gel along with a known amount of a control DNA for comparison. Alternatively, direct measurement using fluorescent dye based kit (e.g., Qubit™) will ensure the proper amount of DNA is submitted.  AB1Size of PCR ampliconng of DNA (in 3 μl sample)2100 bp1 - 123500 bp5 - 6041000 bp10 - 12053000 bp30 - 36065000 bp50 - 600
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is our lab's version of the standard lithium acetate-based transformation protocol originally developed by the Gietz lab (see eg <a href="http://www.nature.com/nprot/journal/v2/n1/abs/nprot.2007.13.html" target="_blank">Gietz and Schiestl 2007</a>).</p>

[BEFORE_START]
<p>Before you start, you will need:</p>
<ul>
<li>A suitable yeast strain to be transformed</li>
<li>A suitable plasmid, with a yeast origin of replication (centromeric or 2μ) and a selectable marker that matches the genome of the yeast strain you are transforming (eg a <em>URA3</em> marker if this gene is non-functioal in your strain's genome). Typical minipreps, which give yields of 50 ng/μl or higher, are suitable. </li>
<li>Suitable selective plates that match the selectable marker on the plasmid (eg standard dropout plates lacking uracil for a <em>URA3</em> marker plasmid)</li>
<li>Suitable medium for growing the non-transformed yeast strain (normally YPD medium, ie 2% glucose, 1% Yeast extract, 2% peptone in water).</li>
</ul>

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

[DESCRIPTION]
<p>Procedimiento basado en la sedimentación a través de una barrera de densidad de 1,077g/ml dejando en la interfase los PBMCs. </p>
<p> </p>
<p>La realizacion de este protocolo fue posible gracias al apoyo del departamento administrativo de ciencia tecnología e innovacion, Colciencias a traves del proyecto 111356933173 convocatoria569-2012.</p>

[BEFORE_START]
<p>Prepare la solución A con materiliales libres de LPS. </p>
<p>Quite el freno de la centrifuga y organice el programa con el que va a trabajar (700 g - 20 minutos - temperatura ambiente - SIN FRENO). </p>
<p> </p>

[GUIDELINES]
<p>Para aislar Celulas Mononucleares de Sangre Periferica (PBMCs), extraer 4 mL de sangre de una persona voluntaria sana. Bajar la densidad de la sangre y sedimentar por centrifugación en una barrea de densidad a1.077g/mL. La intefase que contiene los PBMCs se extrae y se diluye en solucion A 1:1 y se centrifuga a 150 g por 10 minutos; el pellet celular es resuspendido en medio de cultivo (RPMI 1640, Antibiotic antimicotic 1X).</p>
<p> </p>
<p>Las celulas son cultivadas por 12 horas antes del ensayo a 37 ºC y 5 %CO<sub>2. </sub></p>
<p> </p>
<p> </p>

[STEPS]
?. 
?.
TITLE: Cloning of L0 parts into pUAP-ye for Loop typeIIS
AUTHORS: Eftychis Frangedakis, Susana Sauret-Gueto

[STEPS]
?. Design primers as described in the Figure below to add the necessary overhangs for cloning into pUAP-pe and also the overhangs that correspond to the common syntax.
?. DNA parts are PCR amplified from the source DNA part (e.g. plasmid DNA or genomic DNA), using a high fidelity DNA polymerase such as Phusion. Use 10 ng of DNA if the template is plasmid DNA and 100ng if the template is genomic DNA. The cycling conditions are: Denaturation at 98 °C for 30 s. 35 cycles of: denaturation at 98 °C for 10 s, annealing at primer annealing temperature for 30 s, and extension at 72 °C for 15 s/kb. Final extension at 72 °C for 10 min.
?. Run the PCR products on a 1.5% (w/v) agarose gel.
?. Gel extract the band that corresponds to the size of the amplified DNA part using a kit such as QIAquick Gel Extraction Kit.
?. Determine DNA concentration with spectrophotometry (Nanodrop).
?. Prepare aliquots of the DNA part at a concentration of 15 nM and of the pUAP-pe acceptor vector at a concentration of 7.5 nM.
?. Set up a Type IIS assembly reaction into a 0.2 mL tube according to Table  to clone the amplified DNA part into pUAP-pe.  AB1  Sterile water
    5 
  2  pUAP-pe 
    1 
  3  DNA part 
    1 
  4  10x Tango buffer (Thermo Fisher)
    1 
  5  1 mg/mL bovine serum albumin (NEB)
    0.5 
  6  T4 DNA ligase (5 U/µL) (Thermo Fisher)
    0.25 
  7  10mM ATP (SIGMA)
    1 
  8  SapI (LguI) (5 U/µL) (Thermo Fisher)
    0.25 
  9  Final volume
    10
AB1  Sterile water
    5 
  2  pUAP-pe 
    1 
  3  DNA part 
    1 
  4  10x Tango buffer (Thermo Fisher)
    1 
  5  1 mg/mL bovine serum albumin (NEB)
    0.5 
  6  T4 DNA ligase (5 U/µL) (Thermo Fisher)
    0.25 
  7  10mM ATP (SIGMA)
    1 
  8  SapI (LguI) (5 U/µL) (Thermo Fisher)
    0.25 
  9  Final volume
    10
?. Place samples on a thermocycler and use the following program: Assembly: 26 cycles of 37​ °C for 3 min and 16 °C for 4 min. Termination and enzyme denaturation: 50 °C for 5 min and 80 °C for 10 min.
?. Transform 20 μL of chemically competent E. coli cells (transformation efficiency of 1 × 107 transformants/µg plasmid DNA) using 2 μL of the assembly reaction and then spread on LB agar plates containing 25 μg/mL chloramphenicol.
?. Incubate overnight at 37 °C.
?. Select white colonies for sequencing.
?. Confirm the presence of the correct insert with Sanger sequencing using the primers UAP_F and UAP_R and any additional DNA part specific primers.
?.
TITLE: Detection of Equatorial Plasma Bubbles
AUTHORS: swapna.karnam

[DESCRIPTION]
Slant Total Electron Content values from Global Navigation Satellite System are used to detect the Equatorial Plasma Bubbles in the Ionosphere.

[STEPS]
1. Download GPS data from SOPAC website in RINEX format
2. Data is converted from RINEX to .cmn format using GPS TEC software
3. Calculate ROT and ROTI
4. Difference between Evening ROTI and Day time ROTI is calculated
5. If the difference exceeds 0.05, then Equatorial Plasma Bubble is Detected
TITLE: Golden Gate lvl 0
AUTHORS: Vinca Seiler, René Inckemann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Golden Gate reaction protocol for lvl 0</div></div>

[STEPS]
?. [Pipetting scheme for assembly reaction]
of DNA insert ( )
0.5 µl
?. [Pipetting scheme for assembly reaction]
of entry Vector (15 ng/ μL)
0.5 µl
?. [Pipetting scheme for assembly reaction]
T4 DNA Ligase buffer (NEB)
1 µl
?. [Pipetting scheme for assembly reaction]
T4 DNA Ligase (NEB)
0.5 µl
?. [Pipetting scheme for assembly reaction]
Esp3I (NEB)
0.5 µl
?. [Pipetting scheme for assembly reaction]
Water to
10 µl
?. [Thermocycler Rapid Protocol]
37 °C
?. [Thermocycler Rapid Protocol]
16 °C
?. [Thermocycler Rapid Protocol]
Cycle step 8 and 9 5-10x
?. [Thermocycler Rapid Protocol]
37 °C
?. [Thermocycler Rapid Protocol]
50 °C
?. [Thermocycler Rapid Protocol]
80 °C
?. [Transformation]
Add  -  of each assembly reaction to  competent cells.
2 µl
5 µl
50 µl
?. [Transformation]
Cells should be recovered for  (Amp) to  (Kan, Chloramphenicol).
?. [Alternative Thermocycler Improved Protocol ]
37 °C
?. [Alternative Thermocycler Improved Protocol ]
16 °C
?. [Alternative Thermocycler Improved Protocol ]
Cycle step 13 and 14 15x
?. [Alternative Thermocycler Improved Protocol ]
50 °C
?. [Alternative Thermocycler Improved Protocol ]
80 °C