eamag/protocols-with-steps huggingface dataset:
TITLE: Ethanol precipitation of nucleic acid
AUTHORS: Andreas Sagen

[DESCRIPTION]
Simple protocol to precipitate nucleic acids

[STEPS]
SECTION: Sodium acetate (3 M, pH 5.2)
1. Add 25 mL distilled water to a 50 mL canonical tube
SECTION: Precipitation
5. Add 100 µL lysate in a reaction tube
SECTION: Precipitation
6. Add 10 µL Sodium acetate (3 Molarity (M), pH 5.2), 2 µL 250 µg/mL glycogen and 333 µL absolute ethanol

Material:
 
 
SECTION: Precipitation
7. Vortex solution, then precipitate at -20 °C or -80 °C for 60 min or 60 min

 

SECTION: Precipitation
8. Centrifuge at 12000 rcf, 30 min, 4 °C. Discard supernatant, and repeat step 9.1 and 9.2, 2 times in total
SECTION: Precipitation
9.  Air-dry the pellet

 

SECTION: Precipitation
10. Resuspend pellet in 20 µL TE-buffer or nuclease-free water

SECTION: Sodium acetate (3 M, pH 5.2)
4. Autoclave solution at 121 °C for 15 min
SECTION: Sodium acetate (3 M, pH 5.2)
3. Adjust pH to pH 5.2 with glacial acetic acid

Materials:
 
SECTION: Sodium acetate (3 M, pH 5.2)
2. Measure 12.305 g Sodium acetate

Materials:
 
SECTION: Precipitation
8.1. Discard supernatant, and resuspend pellet in 500 µL 70% absolute ethanol
SECTION: Precipitation
8.2. Centrifuge at 12000 rcf, 30 min, 4 °C
TITLE: Qubit dsDNA HS Assay
AUTHORS: Lakme Caceres

[DESCRIPTION]
How to use the Qubit 2.0 fluorometer to quantify dsDNA samples. It is most accurate for sample concentrations from 5 pg/uL to 120 ng/uL.

[GUIDELINES]
Allow reagents to equilibriate to room temperature before use:
HS Buffer (ambient)
HS Reagent (4C - dark)
HS Standard #1 (4C)
HS Standard #2 (4C)

Make sure to use the thin-wall 0.5 mL Qubit PCR tubes for all samples and standards. Only label the caps.

[STEPS]
1. To prepare the Qubit working solution, a mixture of HS Reagent and HS Buffer, take the number of samples you will be running and multiply that by 199 uL. Then add 380 for the HS Standards. Round this number up to the nearest tenth (for excess). This is the amount of HS Buffer you will need. Divide this number by 200 to get the amount of HS Reagent you need to add. 

Example: 
199(8) + 380 = 1,972 --> 1,980 uL
1,980 / 200 = 9.9 uL
Add 9.9 uL HS Reagent to 1,980 uL of HS Buffer to make enough working solution for 8                                      samples and 2 standards.
2. Add 190 uL of working solution to two Qubit tubes. Label the top of the tubes S1 and S2. Then add 10 uL of the respective HS Standard to each tube.
3. For each of your sample tubes, add 199 uL of working solution to labeled tubes. Then add 1 uL of your samples to each tube. 
4. Vortex all tubes and spin down if necessary. Incubate for 2 minutes at room temperature. 
5. On the Qubit, select dsDNA -> dsDNA High Sensitivity -> Read standards
6. Avoid touching the walls of the tube as the Qubit is very temperature sensitive. Insert standard 1 and then select Read standard. Repeat for standard 2. 
7. Select Run samples and read them one by one. Use the 1 uL sample volume. When you are done, select Data at the bottom to view all your sample concentrations and take a photo.
8. To get sample concentrations in ng/uL, multiply Qubit readout by 0.2.
TITLE: Blood Sample collection and PBMC isolation JAGUAR.v4
AUTHORS: Carla Jones, Pablo Romagnoli, Gosia Trynka

[DESCRIPTION]
AIM: This protocol aims to isolate and cryopreserve PBMC from healthy donors from JAGUAR sites for multimodal single cell analysis and blood for DNA extraction for genotyping.

[BEFORE_START]
coolcell must be at room temperature

[STEPS]
SECTION: Blood Collection
1. Collect blood (21g needle) into the tube using the standard technique for CPT tube (3 tubes per donor). After collection, store tubes upright at room temperature (RT, 18-25oC) for up to 2hr (if longer times are required keep tubes on ice until processing).

 

SECTION: Blood Collection
None. 

SECTION: PBMC cryopreservation
9. Centrifuge for 5 min at 300 RCF. Aspirate as much supernatant as possible without disturbing the cell pellet. Resuspend pellets in 500 μl of 100% FCS and transfer all to a cryovial.

SECTION: Blood Collection
2. Collect an extra 1-2mL of blood in an EDTA blood collection tube and transfer blood to a cryotube labeled with a 2D barcode for DNA extraction (whole genome sequencing) and store at -80 until processing
SECTION: PBMC isolation
4. Centrifuge CPT tubes at 1500g RT for 30 minutes (break and acceleration at 5)
SECTION: PBMC isolation
5. Add 2mL washing media to the CPT tube to wash remaining cells and transfer to the same tube above, bring volume to 10 mL. Cap tube. Mix cells by inverting the tube slowly 5 times.

SECTION: PBMC isolation
6. Centrifuge for 5 min at 300 RCF. Tip off supernatant without disturbing the cell pellet.

SECTION: PBMC isolation
7. Add washing media to bring volume to 10 mL. Cap tube. Resuspend cells gently with a pipette
SECTION: PBMC isolation
8. Count cell numbers and viability using Trypan Blue (dilution 1:2) in a Neubauer chamber 
CRITICAL: add 2D barcode stickers to the cryotubes while tube are at room temperature.
SECTION: PBMC cryopreservation
10. Combine 500 μl 2X Freezer media (dropwise) with the cells in cryovial and gently pipette to mix. Place the cryovial into the Mr. Frosty (or CoolCell) and transfer to -80 oC as soon as possible.
SECTION: PBMC cryopreservation
11. CRITICAL: cell viability will be directly correlated to the time cells spend in the presence of DMSO - the quicker the better
SECTION: PBMC cryopreservation
12. After 24 hrs, move cryovials into liquid Nitrogen tanks until shipping, record date of liquid nitrogen transfer
SECTION: PBMC isolation
3. After centrifugation, pipette to remove approximately half of the plasma (stored in a new tube at -80C) without disturbing the cell layer. Collect the cell layer with a Pasteur Pipette and transfer it to a 15 mL Falcon tube with a cap. *Collection of cells immediately following centrifugation will yield the best results.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The workflow applied to Tara Oceans raw sequence data for generating assemblies suitable for genomic binning.</p>
<p> </p>
<p>Used in:</p>
<p>"290 Metagenome-assembled Genomes from the Mediterranean Sea: Ongoing Effort to Generate Genomes from the Tara Oceans Dataset" - bioRxiv https://doi.org/10.1101/069484</p>
<p>"Undocumented potential for primary productivity in a globally-distributed bacterial photoautotroph" - submitted</p>

[STEPS]
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TITLE: Addition of the adaptor to RNA substrates for 5′ RACE
AUTHORS: Matus Valach

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Simple protocol for mapping 5′-P RNA termini by RT-PCR after the addition of a 5′ adaptor using T4 RNA ligase 1.</div></div>

[STEPS]
?. Mix the following components (10 μL):  ABC1ComponentAmount [μL]Final concentration2RNA [1 µg]4100 ng/µL35′ RACE RNA oligo [100 µM]220 µM4RNase-free water (ddH2O)4
ABC1ComponentAmount [μL]Final concentration2RNA [1 µg]4100 ng/µL35′ RACE RNA oligo [100 µM]220 µM4RNase-free water (ddH2O)4
?. Denature for 2 min at 70 °C, place on ice.
?. Mix the following components (20 µL):  ABC1ComponentAmount [μL]Final concentration2RNA + oligo mix (step 2)10310× RNA ligase buffer 21×450% PEG-8000615%5ATP [10 mM]21 mM6T4 RNA ligase 1 [10 U/µL]21 U/µL
ABC1ComponentAmount [μL]Final concentration2RNA + oligo mix (step 2)10310× RNA ligase buffer 21×450% PEG-8000615%5ATP [10 mM]21 mM6T4 RNA ligase 1 [10 U/µL]21 U/µL
?. Incubate using the following program: 60 min at 25 °C, then 60 min at 16 °C, and then for 120 min at 4 °C.
?. Purify the RNA from the RNA ligase reaction (e.g., trizol extraction or column clean-up).
?. Proceed to RT-PCR. For the RT reaction, use 250-500 ng of the purified adaptor-ligated RNA and random hexamers (or a primer of choice). For the PCR, use an upstream (forward) primer binding to the 5′ adaptor and a downstream (reverse) primer binding to the RNA of interest.
TITLE: Culturing Xanthomonas
AUTHORS: Murray  Grant, Shannon  Greer, Joana  Vicente

[DESCRIPTION]
This is the protocol for culturing strains of Xanthomonas species in preparation for extracting genomic DNA for sequencing.

[STEPS]
1. Streak isolates onto King’s B plates. Incubate at 28°C for ~48 hours.
2. Pick a single colony and inoculate to 10ml King’s B liquid media in 30ml universal tubes. Incubate over night at 28°C and 220rpm.   
3. Pellet 1.8ml of liquid culture in a 2ml Eppendorf tube by centrifuging at 5000g for 10mins. Pour off and discard the supernatant.   
4. Pipette another 1.8ml culture into the tube and repeat step 3. This will pellet 4ml of culture in a 2ml Eppendorf tube.   
5. Spin the tubes for 2mins at 5000g. Remove the supernatant by pipetting and discard.   
6. Proceed to the extraction process or flash freeze the pellets in liquid nitrogen and store at -20C until use.
TITLE: Ionpath MIBI (Multiplexed Ion Beam Imaging) Protocols -- University of Minnesota TMCs
AUTHORS: Ionpath Inc.

[DESCRIPTION]
Link to information regarding Ionpath MIBI as used by the University of Minnesota

[STEPS]
1. Links to company sites:
https://www.ionpath.com/
https://www.ionpath.com/mibiscope/ 
2. Slides and Reagents from Ionpath for MIBI 
    Slides (Cat#: 567001): specially formulated, high-purity gold-coated slides 
    Reagents
            MIBI 20X TBS-T (Cat#: 567005) 
            MIBI 10X PBS (Cat#: 567004)
            MIBI 10X Tris pH8.5 (Cat#: 567003)
            MIBI diH2O (Cat#: 567002) 
Current Ionpath company Conjugated Antibody information for human FFPE tissue and mouse FFPE or frozen tissue: https://www.ionpath.com/conjugated-antibodies/
https://www.ionpath.com/antibody-panels/
Ionpath Conjugation kits: 
https://www.ionpath.com/antibody-conjugation-kit/
3. Overview of the MIBI staining protocol with Ionpath reagents and slides
1. Prepare and bake the slides 
2. Deparaffinization and Rehydration 
3. Heat-induced Antigen retrieval
4. Wash with MIBI 20X TBS-T (Cat#: 567005)
5. Blocking
6. Antibody Staining (Metal-conjugated Abs) 
7. Fixation and Dehydration 
8. Dry and Store the slides if not processing on scope right away.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a collection of methods from [tentative citation]:<br /><br /><span style="color: #3c3c39;">Jeff Strohm, Robert Hanner, Richard J Heck. (2018) A metabarcoding perspective on soil biodiversity under different tillage treatments. <em>Submitted to</em> Metabarcoding and Metagenomics</span><span style="color: #3c3c39;"><br /></span></p>

[STEPS]
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span lang="EN-US" style="margin: 0px; line-height: 200%; font-family: 'Arial',sans-serif; font-size: 10pt;">Increasing energy expenditure by stimulating thermogenesis through activation of brown adipose tissue (BAT) and/or induction of browning of white adipose tissue (WAT) is considered a promising strategy to treat/prevent obesity and related metabolic diseases</span><span lang="EN-US" style="margin: 0px; line-height: 200%; font-family: 'Arial',sans-serif; font-size: 10pt;">. Whereas WAT is adapted to store energy as triglycerides, BAT produces heat (non-shivering thermogenesis). In brown adipocytes, the uncoupling protein-1 (UCP-1) regulates conversion of energy into heat by uncoupling ATP production from mitochondrial respiration</span><span lang="EN-US" style="margin: 0px; line-height: 200%; font-family: 'Arial',sans-serif; font-size: 10pt;">. Also in WAT adaptive UCP-1 positive adipocytes (brown in white: brite or beige) can arise, predominantly in subcutaneous (s) WAT</span><span lang="EN-US" style="margin: 0px; line-height: 200%; font-family: 'Arial',sans-serif; font-size: 10pt;">. This browning of WAT is enhanced by exposure to cold temperatures</span><span lang="EN-US" style="margin: 0px; line-height: 200%; font-family: 'Arial',sans-serif; font-size: 10pt;">. </span></p>

[BEFORE_START]
<p>-Turn on 37°C oven<br />-Prewarm slide box and 0.1% CaCl<sub>2</sub> solution</p>
<p>-Make sure TNB is stored at -20°C</p>

[GUIDELINES]
<p>-To dissolve the blocking reagent in TNB, heat TNB blocking buffer to 60°C for 1 hour with stirring. Aliquot over 15 ml tubes. TNB may be stored for up to 1 month at -20°C.<br />-Pre-incubate the secondary goat anti-rabbit-biotin couped antibody with the pre-immune serum 1 day before use.<br />-Harris hemtaoxylin: 5 sec is a guideline. If solution is fresh, it is best to reduce the time, otherwise sections will be overstained. The Harris hematoxylin work solution has to be replaced every two weeks and filtered daily.</p>

[STEPS]
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TITLE: AF &amp; HCM meta-analysis protocol
AUTHORS: Max Aboutorabi

[DESCRIPTION]
Background: A systematic review and meta-analysis will be conducted to assess the efficacy and safety of direct oral anticoagulants (DOACs) versus vitamin K antagonists (VKAs) in patients with hypertrophic cardiomyopathy (HCM) and atrial fibrillation (AF). 

Methods: Studies enrolling patients with HCM and AF will be included. Primary outcomes will include thromboembolic events, major bleeds, and all-cause mortality, while secondary outcomes will include intracranial hemorrhages and cardiovascular death. 

Results: We will search OVID MEDLINE, Cochrane CENTRAL, and EMBASE up to April 2024. Data will be extracted and analysed using standard meta-analytic techniques.

Conclusion: Our findings will provide insights into the comparative effectiveness of DOACs and VKAs in patients with HCM and AF, aiding clinical decision-making in this population.

[STEPS]
SECTION: AF &amp; HCM meta-analysis protocol
1. Identify the question using the PICO framework
Population - patients with concurrent AF and HCM (No constraints were set in terms of type of atrial fibrillation or type of DOAC or VKA the patient was given. Furthermore, there were no constraints in terms of type of hypertrophic cardiomyopathy or duration of the condition)
Intervention - DOACs
Comparison - Vitamin K antagonists
Outcomes - Rates of thromboembolic events, major bleeds, all-cause deaths, intracranial haemorrhages, and cardiovascular deaths

SECTION: AF &amp; HCM meta-analysis protocol
2. Search strategy
Databases to search include OVID MEDLINE, Cochrane CENTRAL and EMBASE, from inception to April 2024. 
Search terms included atrial fibrillation, hypertrophic cardiomyopathy, warfarin, anticoagulation, Vitamin K antagonist, and direct oral anticoagulant, and includeMeSH terms, and keywords. 
Two reviewers will independently screen for titles for relevance and then assessed the full texts for their eligibility. 
A third expert will intervene whenever discrepancies are observed.
SECTION: AF &amp; HCM meta-analysis protocol
3. Data extraction
The data will be extracted onto a pre-made excel spreadsheet (Office 365, Microsoft Corporation) with subheadings for study type, single or multi-centre, study drugs, and population characteristics: age, sex, hypertension, diabetes, heart failure, left atrium (LA) size, left ventricular ejection fraction (LVEF), maximal wall thickness, left ventricular outflow tract (LVOT) gradient, and follow-up duration.
SECTION: AF &amp; HCM meta-analysis protocol
4. Data analysis
The data will be analysed using odds ratios as a measure of effect. A random-effects model will be used for accounting for the observed heterogeneity. Statistical analysed and creation of forest plots will be completed using meta packages in R programme and variance will be calculated using paule-mandel model.
SECTION: AF &amp; HCM meta-analysis protocol
5. Risk of bias Assessment
The studies included will be assessed by 2 independent authors and will use the Newcastle Ottawa scale.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is a protocol from: <br /><br />Stedman, K. M., K. Porter, and M. L. Dyall-Smith. 2010. Chapter 6: The isolation of viruses infecting Archaea. Manual of Aquatic Viral Ecology. Waco, TX:American Society of Limnology and Oceanography. doi:10.4319/mave.2010.978-0-9845591-0-7<br /><br />Please see the <a href="http://www.aslo.org/books/mave/MAVE_057.pdf" target="_blank">published manuscript</a> for additional information.

[GUIDELINES]
<strong>Authors: </strong>Kenneth M. Stedman<sup>1</sup>, Kate Porter<sup>2</sup>, and Mike L. Dyall-Smith<sup>3</sup> <br /><sup>1</sup>Department of Biology, Center for Life in Extreme Environments, Portland State University, P.O. Box 751, Portland, OR 97207, USA <br /><sup>2</sup>Biota Holdings Limited, 10/585 Blackburn Road, Notting Hill Victoria 3168, Australia <br /><sup>3</sup>Max Planck Institute of Biochemistry, Department of Membrane Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany<strong><br /></strong><em><br /></em>Standard negative stain EM works best on samples with low salt concentrations, but many haloviruses are stable only at high salt concentrations. If one uses high salt preparations, the salts can crystallize on the grid, occluding the particles and heating up the specimen. One way to overcome the problem is to first fix the sample using gluteraldehyde. Another problem is poor adsorption to plastic-coated grids (e.g., formvar). Pretreatment of the grids with poly-L-lysine can alleviate this issue. <br /><br />The method for examining haloviruses was adapted from that described by Tarasov et al. (2000).<em><br /><br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/23c2xe.png" alt="" data-src="https://s3.amazonaws.com/pr-journal/23c2xe.png" data-ofn="MAVE ch 6 fig 3.png" /><br /></em><strong>Fig. 3:</strong> Electron micrographs of haloviruses and VLPs. <br />(A) Spindle-shaped particles of His1 virus (host is <em>Har. hispanica</em>). <br />(B) Spherical particles of SH1 virus. Also seen is a flagellar filament from the host (<em>Har. hispanica</em>). <br />(C) Head-tail VLPs, and other structures, seen in a natural hypersaline water sample (Serpentine lake, Rottnest Island, Western Australia). All scale bars 200 nm. Negative stain with uranyl acetate.<em> <br /></em>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is a technique for isolating phage from agar plates.&nbsp; When working with lytic phage, well-isolated, cleared areas in a lawn of bacteria (called plaques) are removed from the agar (by coring with a sterile pipette) and used to infect fresh bacteria in liquid medium.&nbsp; The lysed culture can be used for a new plaque assay after 0.2 um filtering to remove residual bacteria.&nbsp; New phage are "plaque-purified" a minimum of three times before they are considered to be free of other phage.

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol provides an inexpensive and fast way to cleanse the supernatant of <em>Giardia</em> trophozoites' culture from bovine proteins and any other protein digests present in TYI-S-33. </p>

[BEFORE_START]
<p>Warm non-supplemented DMD+Pen/Strep (1%) and 1xPBS to 37°C. </p>

[STEPS]
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TITLE: Interventions (Part 5 of &#34;Effects of Online Exercise Intervention on Physical and Mental Conditions in Young Adults with Chronic Neck Pain&#34;)
AUTHORS: Yiting Lin

[DESCRIPTION]
This is Part 5 "Effects of Online Exercise Intervention on Physical and Mental Conditions in Young Adults with Chronic Neck Pain".
Participants in both groups completed the same exercise program three times a week for six weeks with either face-to-face or online mode of delivery by physiotherapists. The intervention includes:
A. Warm-up exercises
B. Cranio-cervical flexion exercises
C. Strength-endurance exercises
D. Scapular stabilization exercises
E. Stretching exercises

[GUIDELINES]
Interventions:

Participants in both groups completed the same exercise program three times a week for six weeks with either face-to-face or online mode of delivery by physiotherapists.

At baseline, the suitable elastic bands (Thera-band®) for performing the exercises were selected by testing the 15 repetitions maximum (15RM) of the Modified Brügger’s Exercise (MBE) and the Modified Proprioceptive Neuromuscular Facilitation Diagonal Flexion Exercise (MPNFDFE) for the participants of both the experimental group and control group. The details of the MBE and MPNFDFE were described in supplementary file 1.

Participants randomized to the control group performed the following exercises three times a week for six weeks in a group of 3-5 participants in the laboratory of the Sport Medicine and Rehabilitation School of the Beijing Sport University:

A. Warm-up exercises
B. Cranio-cervical flexion exercises
C. Strength-endurance exercises
D. Scapular stabilization exercises
E. Stretching exercises

The details of the exercises were described in supplementary file 2.

Participants allocated to the experimental group performed the same set of exercise three times a week for six weeks in their home, with the following arrangements:

The participants had weekly online exercise meetings with the physiotherapist in a group of 3-5 participants using the TENCENT Meeting. Then the participants performed the remaining two sessions of exercises themselves weekly. If they had encountered any problems, they could consult the physiotherapist during the online session or texting to the physiotherapist using the WeChat app. All the participants of the experimental group received a package of exercise pamphlet and a video disc containing 17-minute exercise demonstrations by the physiotherapist.

[STEPS]
TITLE: Generation of CRISPR constructs
AUTHORS: Thanh Ngoc Nguyen

[DESCRIPTION]
This protocol details the procedure of generation of CRISPR constructs.

[STEPS]
SECTION: Procedure
1. Designing gRNAs using https://chopchop.cbu.uib.no.
 

SECTION: Procedure
2. Choose the top-ranking gRNA sequences that target the earliest exon possible.
 

SECTION: Procedure
3. Click on the chosen target sequence, another window with all the information related to this gRNA sequence will appear.
 

SECTION: Procedure
4. Copy the target sequence without the PAM into the highlighted region of the below sequence:
 

SECTION: Procedure
1.1. “Target”: Put in the gene name/“In”: choose the species.
 

SECTION: Procedure
1.2. Do not change anything in “General” tab.
 

SECTION: Procedure
1.3. In the “Cas9” tab, make sure you choose “No requirements” for “5’ requirements for sgRNA” and tick “I intend to replace the leading nucleotides with “GG”” (3 options of the “Sef-complementarity (Thyme et al.)” should be ticked).


SECTION: Procedure
1.5. Click “Find target sites”.
SECTION: Procedure
1.4. In “Primers” tab, I choose product size from 200 to 500 and minimum distance from primer to target site at least 100.

SECTION: Procedure
5. Order the above sequence as a primer for Gibson assembly.
SECTION: Procedure
6. Preparing cut pSpCas9(BB)-2A-GFP:

SECTION: Procedure
7. Dilute the primer from steps 4 and 5 to the final concentration of 0.8 micromolar (µM) (1/125 dilution of the 100 micromolar (µM) stock). Set up a Gibson assembly reaction as following:
1 µL of the diluted primers
1 µL of BbsI-linearised pSpCas9(BB)-2A-GFP
2 µL of HiFi DNA Assembly Master Mix
Incubate at 50 °C for 120 min.

SECTION: Procedure
8. Transform 1.8 µL of the mix from step 7 (the rest can be stored at -20 °C as a backup in case the transformation does not result in any colonies) using 10 µL of the NEB® 5-alpha Competent E. coli cells with manufacturer’s instructions. 
 

SECTION: Procedure
9. The next day, pick up a few colonies and set up overnight cultures in growth broth.
SECTION: Procedure
10. Miniprep the cultures to purify plasmids and send them for sequencing using this primer (5’ GCTCACCTCGACCATGGTAAT 3’).
SECTION: Procedure
11. Once sequenced verified, the CRISPR constructs are now ready to be used for transfection.
SECTION: Procedure
6.1. Cut the vector with BbsI:
       10 µg of vectors
       2 µL of BbsI
       3 µL of NEBuffer™ r1.1
       Add sterile milliQ water to 30 µL
       Incubate for 6-8 hours at 37 °C
SECTION: Procedure
6.4. Run the reaction on a 0.5 % DNA agarose gel.
SECTION: Procedure
6.3. Heat de-activate at 60 °C for 5 min.
SECTION: Procedure
6.2. After that, add 1 µL of CIP and incubate for no longer than 60 min.
SECTION: Procedure
6.5. Extract the cut vector, determine the concentration, dilute it to 10 ng/µl and aliquot to 1 µL aliquots and store at -20 °C.