eamag/protocols-with-steps huggingface dataset:
TITLE: Acropora DNA extraction with Qiagen DNeasy tissue kit
AUTHORS: Iliana Baums, Sheila Kitchen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>DNA extraction protocol for Acropora or other coral tissue based on Qiagen DNAeasy kit. This extraction protocol works well for the Acropora SNPchip and other coral genotyping applications (such as microsatellite genotyping). It preferrentially extracts coral host DNA but some </span><span style = "font-style:italic;">Symbiodiniacea</span><span> DNA will be present. </span></div></div>

[STEPS]
?. [Sample preparation]
Use sterile technique to cut 3-4 polyps (skeleton with tissue) off the coral fragment with a razor blade.
?. [Sample preparation]
Transfer the pieces to a 1.5 ml tube/96-well plate.
?. [Sample preparation]
Add 180 ul of Buffer ATL and 20 ul of proteinase K (from Qiagen kit) to each tube/well.
?. [Sample preparation]
If using the tubes, invert the tubes several times to gently mix the reagents. *Do not vortex*
?. [Sample preparation]
Incubate the mixture overnight at 56 °C.
?. [Sample preparation]
a.The mixture will have a slight yellow hue after lysis from the algal symbionts.
?. [Sample preparation]
b.The tubes or plate can be placed on a rocking platform for gentle agitation.
?. [Qiagen DNeasy Blood and Tissue Kit protocol]
Follow Qiagen’s protocol with the following changes:Following step 6 of the spin-column protocol, we perform the optional centrifugation for 1 min at 20,000 xg to dry the column membrane.
?. [Elution 2x]
Elution 1- Add 100 ul of low TE buffer directly to the column membrane, incubate for 1 min and centrifuge as recommended. a.This elution typically contains smaller fragmented DNA.
?. [Elution 2x]
Elution 2 – Move the column to a new 1.5 ml tube. Repeat elution as above, but increase incubation of 100 ul of low TE to 5 min. a.This elution contains high molecular weight DNA and is used for downstream analyses. b.Store at -20 °C.
?. [Check extraction quality]
Run DNA on a 1.5% agarose gel to check extraction quality of elution 1 and 2. Look for one high molecular weight band at the top of the gel with little smearing.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is a standard polymerase chain reaction, optimized to amplify a () bp gene flanked by two loxP sites. (?)</p>
<p> </p>
<p>Refer to page 32, April 5, 2017 entry in Miguel's laboratory notebook for the original document.</p>
<p> </p>

[STEPS]
?. 
?. 
?.
TITLE: ITS and LSU nrDNA analyses
AUTHORS: Maria P Martin, Thiago Accioly, Julieth O. Sousa, Melanie Roy , Monique Gardes, Iuri G. Baseia

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">After DNA extraction, the ITS regions were amplified and sequenced using the primer pair ITS1F/ITS4. Part of the LSU was amplified and sequenced using either the primer pair LR0R/LR5. When amplifications were not obtained, nested PCR of ITS and semi-nested PCR of LSU were performed. The samples were sequenced bidirectionally in Macrogen Inc. (Seoul, South Korea). </div><div class = "text-block">The molecular dataset of unambiguously aligned ITS sequences was submitted to three types of analyses: maximum parsimony (MP), maximum likelihood (ML), and Bayesian inference. Concatenate ITS/LSU analyses were also carried out through each method, but concatenated datasets included only those specimens which have sequences for both ITS and LSU nrDNA </div></div>

[STEPS]
?. 
?. 
?. 
?.
TITLE: LC-MS/MS Label-Free Proteomic Data Analysis Parameters
AUTHORS: Danielle Gutierrez, Jamie Allen, Zach Jenkins, Jeff Spraggins

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">List of parameters and settings for searching label free proteomic data in MaxQuant version 1.6.17.</div></div>

[STEPS]
?. Label-free proteomic samples were searched using MaxQuant version 1.6.17.
?. Group Specific Parameter settings included: StandardMultiplicity = 1Variable modifications: Oxidation (M); Acetylation (Protein N-term), Carbamidomethyl (C)Fixed modifications: Carbamidomethyl (C)Max number of modifications per peptide: 5Instrument settings: Orbitrap (default settings)Digestion: Specific, Trypsin /PMax missed cleavages: 2LFQ: None
?. Global Parameters included:Database: UniProt Human Proteome (UP000005640, 9606) Reviewed, downloaded July 30, 2019Min peptide length: 7Max peptide mass: 4600 DaMS/MS analyzer: Default settingsIdentification: Default settings
TITLE: SDS-PAGE and western blot analysis
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol describes SDS-PAGE and western blot analysis.

[STEPS]
SECTION: SDS-PAGE and western blot analysis
1. For SDS-PAGE and western blot analysis, collect cells by trypsinization and subsequent centrifugation at 300 x g, 5 min, 4 °C. 

SECTION: SDS-PAGE and western blot analysis
2.  Wash the cell pellets in PBS and centrifuge once more at 300 x g, 5 min, 4 °C.

SECTION: SDS-PAGE and western blot analysis
3. Remove the supernatant and lyse the cell pellets in RIPA buffer supplemented by cOmplete EDTA-free protease inhibitors (11836170001, Roche) and phosphatase inhibitors (Phospho-STOP, 4906837001, Roche).
SECTION: SDS-PAGE and western blot analysis
4. After incubating in RIPA buffer for 20 min on ice, clear the samples by centrifugation at 20000 x g, 10 min, 4 °C.
SECTION: SDS-PAGE and western blot analysis
5. Collect the soluble supernatant fraction and measure the protein concentrations using the Pierce Detergent Compatible Bradford Assay Kit (23246, Thermo Fisher). 
SECTION: SDS-PAGE and western blot analysis
6. Then, adjust the samples for equal loading and mix it with 6x protein loading dye, supplemented with 100 mM DTT. Then, boil it for 5 min at 95 °C.
SECTION: SDS-PAGE and western blot analysis
7. Load the samples on 4-12% SDS-PAGE gels (NP0321BOX, NP0322BOX, or NP0323BOX, Thermo Fisher) with PageRuler Prestained protein marker (Thermo Fisher).
SECTION: SDS-PAGE and western blot analysis
8. Transfer the proteins onto nitrocellulose membranes (RPN132D, GE Healthcare) for 60 min at 4 °C using the Mini Trans-Blot Cell (Bio-Rad).
SECTION: SDS-PAGE and western blot analysis
9. After the transfer, block the membranes with 5% milk powder dissolved in PBS-Tween (0.1% Tween 20) for 60 min at Room temperature.
SECTION: SDS-PAGE and western blot analysis
10. Incubate the membranes 60 min at 4 °C with primary antibodies dissolved in the blocking buffer and wash.

SECTION: SDS-PAGE and western blot analysis
10.1. Wash for 5 min.(1/3)

SECTION: SDS-PAGE and western blot analysis
10.2. Wash for 5 min.(2/3)
SECTION: SDS-PAGE and western blot analysis
10.3. Wash for 5 min.(3/3)
SECTION: SDS-PAGE and western blot analysis
10.4. Then, incubate with species-matched secondary horseradish peroxidase (HRP)-coupled antibodies diluted 1:10,000 in blocking buffer for 60 min at Room temperature.
SECTION: SDS-PAGE and western blot analysis
11. Afterwards wash the membranes three times with PBS-T and further process for western blot detection.
SECTION: SDS-PAGE and western blot analysis
12. Incubate the membranes with SuperSignal West Femto Maximum Sensitivity Substrate (34096, Thermo Fisher) and image it with a ChemiDoc MP system (Bio-Rad).
SECTION: SDS-PAGE and western blot analysis
13. Analyze the images with ImageJ [57].
TITLE: Thermolabile Proteinase K Typical Reaction Protocol
AUTHORS: New  England   Biolabs

[DESCRIPTION]
Thermolabile Proteinase K is an engineered, subtilisin-related serine protease that will hydrolyze a variety of peptide bonds and is frequently used to cleanup enzymatic reactions or cell lysates.
Heat inactivated following incubation at 55°C for 10 minutes.
Optimal activity and stability for up to 24 months.
Active in a wide range of reaction buffers with optimal activity between 20 - 40°C and pH 7.0 - 9.5. 
No detectable endonuclease, exonuclease, DNase or RNase contaminating activities.

[STEPS]
1. Reactions may be scaled-up linearly to accommodate larger amounts of substrate and larger reaction volumes. Optimal buffering reagents, enzyme quantity, incubation temperatures and times may vary for particular substrates. Typical restriction enzyme cleanup conditions are as follows:

To a 50 µL containing 1 µg and 10 units of a restriction enzyme: 
2. Add 1 µL, mix gently.
3. Incubate reaction at 37 °C for 15 min.
4. Incubate reaction at 55 °C for 10 min to inactivate Thermolabile Proteinase K.
TITLE: Expression and purification protocol of PI3KC3-C1 complex
AUTHORS: Chunmei Chang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines the procedure for expression and purification of the PI3KC3-C1 complex.</div></div>

[STEPS]
?. [Plasmid amplification]
Amplify the plasmid.
constructs: pCAG-VPS15, pCAG-TSF-VPS34, pCAG-TSF-BECN1, pCAG-GST-TEVs- A TG14All of the constructs are Amp resistant and high copy, could be amplified in normal XL10 strain.
?. [Transfection in HEK GnTI cells]
Grow HEK293 GnTI suspension cells to 1.5-2 million cells/ml in the desired volume,  would be good for PI3KC3-C1 complex.
[cells]
?. [Transfection in HEK GnTI cells]
In warm hybridoma media, make 20 X solution of DNA () and PEI ().
i.e. For a 1 L transfection add   to   and   in another  . The ratio of the DNA is: 1.5 ATG14: 1.0 VPS34: 1.0 VPS15: 1.0 BECN1.
?. [Transfection in HEK GnTI cells]
Add PEI to the DNA, mix together and incubate for a further .
?. [Transfection in HEK GnTI cells]
Add the DNA and PEI mixture to cells.
?. [Harvest cells]
Harvest cells after expression for  - .
?. [Harvest cells]
Spin down cells at .
Centrifuge: 2000 33, 20 min, 4 10
?. [Harvest cells]
Wash the pellet with 1 X PBS once, freeze and store in .
-80 °C
?. [Protein purification]
Lyse cells with the lysis buffer ( for ) supplemented with protease inhibitors. Clarify the lysate by centrifugation ().
[lysis buffer]
[cell pellet]
Centrifuge: 16000 33, 60 min, 4 10
?. [Protein purification]
Incubate the supernatant with  at  (or ) for .
[Glutathione Sepharose 4B]
4 °C
4 Room temperature
?. [Protein purification]
Apply the mixture to a gravity flow column and wash the resin extensively with wash buffer.
?. [Protein purification]
Elute the protein complex with .
[GSH elution buffer]
?. [Protein purification]
Add  to eluted proteins and incubate at .
[TEV protease]
4 °C
?. [Protein purification]
Apply the TEV-treated complexes to a 1 ml Strep-Tactin Sepharose gravity flow column, wash the resin extensively with wash buffer.
?. [Protein purification]
Elute the protein complex with .  per fraction.
[Strep elution buffer]
1 mL
?. [Protein purification]
Apply the eluted fraction with highest concentration (or gently concentrate all the fractions to ) to Superdex 6 column (10/300 Increase).
1 mL
?. [Protein purification]
Collect the peak fractions and use freshly.
TITLE: Gentle Cell extraction from rock samples.
AUTHORS: Jackie Goordial, Beth Orcutt, Tim Dangelo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Method for extracting cells in a gentle manner from rocks for downstream single cell sorting and sequencing. Starting material for the protocol is rock in a 15 mL centrifuge tube, stored frozen in 5 mL of Glycerol TE. GlyTE stock recipe:</div><div class = "text-block">1. Mix the following:</div><div class = "text-block">a. 20 mL 100x TE pH 8.0</div><div class = "text-block">b. 60 mL deionized water</div><div class = "text-block">c. 100 mL molecular-grade glycerol (use a syringe)</div><div class = "text-block">2. Pass the glyTE stock through a 0.2 micrometer pore size filter</div><div class = "text-block">3. Store at -20ºC.</div></div>

[STEPS]
?. [Sample is started from 5 g of rock, preserved in 5 mL of glycerol TE in a 15 mL centrifuge tube. ]
a. For one tube (for later sorting) Resuspend the pellet in 1ml of 1X GlyTE. Can dilute with 1X PBS. Store in freezer.  b. For second tube (for microscopy) – stain with SYBR Green, and filter on 0.2 um polycarbonate filter, rinse and dry filter, then mount on glass slide with Citifluor antifade mounting solution. Count cells, or store slide at 4C until ready to count cells
?. [Sample is started from 5 g of rock, preserved in 5 mL of glycerol TE in a 15 mL centrifuge tube. ]
Add EDTA to 15 mL tube ( containing 5 mL glyTE and 5 g crushed rock), add EDTA for final concentration of 10 mM.
?. [Sample is started from 5 g of rock, preserved in 5 mL of glycerol TE in a 15 mL centrifuge tube. ]
Vortex two tubes at time horizontally in tube holder of the Vortex-Genie-2. Vortex for 5 min at speed 7 (too high of a speed could allow the rock particles to cause damage to the cells)
?. [Sample is started from 5 g of rock, preserved in 5 mL of glycerol TE in a 15 mL centrifuge tube. ]
Sonicate, two tubes at a time in ice water, for 1 Minute
?. [Sample is started from 5 g of rock, preserved in 5 mL of glycerol TE in a 15 mL centrifuge tube. ]
Centrifuge at 1,000 RPM for 5 minutes in the bench-top centrifuge in the Emerson Lab to pellet rock particles. Transfer the supernatant to a fresh 15 ml Falcon tube
?. [Sample is started from 5 g of rock, preserved in 5 mL of glycerol TE in a 15 mL centrifuge tube. ]
Centrifuge the supernatant 2ml at time @ 10,000 RPM for 5 minutes in 2 x 2 ml centrifuge tubes. One tube for sorting, and another as replicate or microscopy. Approximately 2.5 ml will be pelleted in each tube over two rounds of centrifugation (2 ml, 2ml, ~ 250 ul). Remove supernatant each time, with caution, using a pipette. Do Not Pour Off Supernatant, cellular material is not always well pelleted and can be lost easily.
TITLE: 2: User-friendly protocol: Oligo ordering and preparation (SABER-FISH)
AUTHORS: Jocelyn Y. Kishi, Sylvain W. Lapan, Brian J Beliveau, Emma R. West, Allen Zhu, Hiroshi M. Sasaki, Sinem Saka, Yu Wang, Constance L Cepko, Peng Yin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is about oligo ordering and preparation.</div></div>

[STEPS]
?. To generate a catalytic (telomerase-like) hairpin corresponding to the primers sequence (RC = reverse complement):A(primer sequence)GGGCCTTTTGGCCC(RC of primer sequence)T(RC of primer sequence)/3InvdT/For example, given the primer 27 sequence of CATCATCAT, the catalytic hairpin h.27.27 sequence would be:ACATCATCATGGGCCTTTTGGCCCATGATGATGTATGATGATG/3InvdT/
?. The /3InvdT/ can be replaced with TTTTTTT for most hairpins to save cost. We have found empirically however, that some hairpins (including those for primers 25, 32, and 41 seem to require the InvdT modification).
?. To change the primer appended to a probe set, a hairpin for primer switching (‘re-mapping’) can be used (Fig. S1a-c). Remapping hairpin is introduced in the same reaction as the catalytic hairpin, and the concentration is flexible (-). Reactions involving primer switching may require additional time for extension to equivalent lengths.To generate a hairpin for primer switching: A(new primer)GGGCCTTTTGGCCC(RC of new primer)T(RT of old primer sequence)/3InvdT/ A detailed schematic for designing such a hairpin can be seen in Fig. S1c. Here, as above, /3InvdT/ can generally be replaced with TTTTTTT for most hairpins to save cost.
?. Fluorescent oligos are designed as follows: /5dye/TT(RC of primer sequence)T(RC of primer sequence)TThis represents a 20mer binding sequence with a 5’ conjugated dye linked to the binding region by a TT’ linker
?. [PER primer re-mapping]
Probe sets with one primer (e.g. primer sequence a) that have to be used in the same experiment with another set that has the same primer (a) can be re-mapped to alternate PER concatemer sequences using a two-hairpin reaction (Fig. S1a-b). An example for designing re-mapping hairpins given a starting primer sequence and desired concatemer sequence is depicted in Fig. S1c. (The original 9 nt primer sequence is not predicted to hybridize to the complementary 20mer imager sequence at 37°C in 1×PBS, so one of the probe sets can usually be concatemerized with the original primer sequence. In this case it is good to have a control condition with just that probe set missing to verify no cross-talk between channels.)
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The ultra-high density intensive farming model of grass carp (<em>Ctenopharyngodon idellus</em>) may elicit growth inhibition, decline flesh quality and increase disease susceptibility of fish. The quality degradation and excessive fat accumulation in cultured <em>C. idellus</em> have long been attributed to possible alterations in the lipid metabolism of fish muscle tissues as a result of over-nutrition from artificial diets. To investigate the effects of different diets on fish muscle quality, a large-scale metabolomics study was performed on 250 tails of <em>C. idellus. </em></p>
<p>The experimental fish were divided into four groups based on sex and diets - female artificial feed (FAF), female grass feed (FGF), male artificial feed (MAF) and male grass feed (MGF). After 113 days rearing period, the AF group showed significantly higher total mass of muscle fat (P &lt; 0.01), with the FAF group being the highest. Metabolomics profiling based on liquid chromatography-mass spectrometry (LC-MS) revealed distinctive patterns of clustering according to the four groups. Overall, artificial feeding was associated with higher concentrations of docosapentaenoic acid (DPA), dihomo-gamma-linolenic acid (DGLA) and arachidonic acid (ARA); whereas grass-feeding was associated with elevated n-3 unsaturated fatty acids (n-3 UFAs), such as eicosapentaenoic acid (EPA), alpha-linolenic acid (ALA) and gamma-linolenic acid (GLA). Artificial feeding also resulted in significant increased docosahexaenoic acid (DHA) in MAF muscle than MGF fish, whereas no significance in the comparison of female samples. Metabolic pathway analyses using both targeted and untargeted approaches consistently revealed that the arachidonic acid metabolism and steroid hormone biosynthesis pathways were significantly different between AF and GF groups.</p>
<p>Our results suggested that grass is a better source of diet fatty acid and protein when compared to artificial feed. Grass feeding could effectively lower triglycerides in serum, reduce fat accumulation and alter lipid compositions in fish muscle by increasing the concentrations of n-3 UFAs, leading to better nutrition and health.</p>

[STEPS]
TITLE: Piezo proteins: incidence and abundance in the enteric nervous system with immunohistochemical techniques
AUTHORS: Michael Schemann, Gemma Mazzuoli-Weber

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is to quantify the presence of both Piezo1 and 2 in enteric neurons throughout the gastrointestinal tract using immunohistochemistry on human samples that were obtained from patients undergoing surgery. The samples were taken from macroscopically unaffected areas. Using immunohistochemical techniques to determine the expression of Piezo channels in the ENS we investigated the co-expression of Piezo1 and 2 channels with a wide range of markers for enteric neuronal populations.</div></div>

[STEPS]
?. Human samples of large and small intestines were taken from macroscopically unaffected areas and placed in an ice-cold-oxygenated sterile Krebs solution and immediately transferred to the laboratory. Tissues were then dissected in the ice-cold oxygenated sterile Krebs solution to obtain whole-mount submucosal and myenteric plexus preparations.
?. Tissue specimens were fixed overnight at room temperature in a solution containing 4% paraformaldehyde and 0.002% picric acid in 0.1 mol/l phosphate buffer and then washed (3 × 10 min) in phosphate buffer.
?. The preparations were then incubated in phosphate-buffered saline (PBS)/ NaN3 (0.1%)/horse serum (HS, 4%) for 1 h at room temperature followed by 48 h and 12 h incubation with the primary and secondary antibody, respectively. For the Piezo1 antibody, double and triple staining for the co-localization study was performed.
?. Specimens were washed in PBS, mounted on poly-l-lysine- coated slides and cover slipped with a solution of PBS (pH 7.0)/NaN3 (0.1) containing 65% glycerol.
?. The preparations were examined with an epifluorescence microscope (Olympus) equipped with appropriate filter blocks. Pictures were acquired with a video camera connected to a computer and controlled by Scion image software (Scion Corp., Frederick, MD, USA). Frame integration and contrast enhancement were employed for image processing.
?. Neurons stained with the antibodies were manually counted and picture of the stained ganglia were acquired.
TITLE: Immunoprecipitation from transfected cells
AUTHORS: Cesare Orlandi

[DESCRIPTION]
This protocol provides step by step information on how to perform immunoprecipitation from transfected cells. We normally use this protocol with HEK293FT cells but it can be adopted to other cell types.

[GUIDELINES]
Note: All solutions should be ice cold and procedures should be carried out at 4°C or on ice.

[STEPS]
SECTION: Immunoprecipitation from transfected cells
1. Drain medium and gently wash cells once with ice-cold PBS (0.5ml for mw6; 1 ml for 6-cm dish; 3 ml for 10-cm dish).
SECTION: Immunoprecipitation from transfected cells
2. Use a gentle stream to dislodge cells with PBS.
SECTION: Immunoprecipitation from transfected cells
3. Centrifuge the tube at 3000 x g for 1 min and discard the supernatant.
SECTION: Immunoprecipitation from transfected cells
4. Resuspend pellet of cells with 500 μl of ice-cold IP buffer
SECTION: Immunoprecipitation from transfected cells
5. Lyse the cells by sonication: 3 times 10’’/30% on ice, waiting 1 minute in between for the lysate to cool down
SECTION: Immunoprecipitation from transfected cells
6. Incubate the whole cell lysate on an end-to-end rocker at 4°C/30’ to solubilize the membrane proteins.
SECTION: Immunoprecipitation from transfected cells
7. Spin down at 14,000 x g for 10 minutes at 4°C to pellet debris and genomic DNA, save the supernatant.
SECTION: Immunoprecipitation from transfected cells
8. Save 75 µl, Cell Lysate (CL) and add 25 µl of SB4X
SECTION: Immunoprecipitation from transfected cells
9. Mix total proteins (equal amounts for each sample) with 10-20 µl of Protein G beads and 2 µg of specific antibody. Note: wash Protein G Beads 3 times with PBS to remove ethanol present in the storage buffer. Let equilibrate in IP buffer for minute before use.
18. 
10. Incubate on an end-to-end rocker at 4°C/overnight
11. Separate the beads and save 75 µl of Flow Through (FT) and add 2 µl of SB4X
12. 10.   Wash beads 3 times with 1 ml of IP buffer
13. 11.   Elute with 50 µl of SB1X (IP)
14. 12.   Warm samples at 42°C/10’ (membrane proteins would start to aggregate at higher temperature).
15. 13.   Load 10 µl of each sample per well for SDS-PAGE
TITLE: Double digested
AUTHORS: Jiaxin Li

[STEPS]
?. Add the following reagents to a tube.  AB1Total10ul210&nbsp;x&nbsp;Buffer1ul3Template（500ng）According to different template4EnzymeEach for 0.5ul5ddWaterAdd to 10ul
AB1Total10ul210&nbsp;x&nbsp;Buffer1ul3Template（500ng）According to different template4EnzymeEach for 0.5ul5ddWaterAdd to 10ul
?. Pipette up and down thoroughly.
?. Put at 37℃ for about 2 hours (according to different enzymes).
?. Agarose Gel Electrophoresis  ABCDEF1GroupDouble restriction digestSingle restriction digest&nbsp;1Single restriction digest&nbsp;2TemplateMarker2Enzyme-digested product10ul10ul10ul3ul（c）/3Theoretical&nbsp;Loading&nbsp;Buffer1.1ul1.1ul1.1ul0.4ul（c）/4Actual&nbsp;Loading&nbsp;Buffer2ul2ul2ul1ul/5Total&nbsp;12ul12ul12ul4ul4ul
ABCDEF1GroupDouble restriction digestSingle restriction digest&nbsp;1Single restriction digest&nbsp;2TemplateMarker2Enzyme-digested product10ul10ul10ul3ul（c）/3Theoretical&nbsp;Loading&nbsp;Buffer1.1ul1.1ul1.1ul0.4ul（c）/4Actual&nbsp;Loading&nbsp;Buffer2ul2ul2ul1ul/5Total&nbsp;12ul12ul12ul4ul4ul