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  6. Protocols for Molecular Biology, Plasmid Cloning, and Viral Preps

    Type
    Protocol
    ...Watch the Video! How to Design a Primer Key considerations when designing primers Watch the Video! Sequence...protocols that you can use for a wide range of applications, with videos for select protocols in the right-hand...right-hand column. You can find even more video content on the Addgene Video Page . We hope that you'll find these...BSL-2 Watch the Video! Water Baths Proper water bath maintenance and use Watch the Video! Pipetting Learn...Link to Video Making LB Agar Plates Create plates to culture bacteria in the lab Watch the Video! Over-... the Video! Streaking Bacteria Isolate single bacterial colonies on an agar plate Watch the Video! Inoculating... the Video! Polymerase Chain Reaction (PCR) Basic PCR protocol with tips and FAQ Watch the Video! Purifying...

  7. Lab Safety for Biosafety Levels One and Two

    Type
    Protocol
    ...These lab safety guidelines provide steps to ensure you are working in BSL-1 and BSL-2 labs safely...., and exposures to hazardous materials. Video Watch the video to learn more about safety in the lab. Equipment...container Fire blanket Fire extinguisher Guidelines BSL-1 Guidelines Before You Work Right after entering ...chemical hygiene officer. BSL-2 Guidelines Remember, the BSL-1 laboratory guidelines above are expected to be...Intro to the Lab Bench Check out more protocols and videos to help you get started in the lab! Introduction...There are four biosafety levels. This protocol provides information for both biosafety level 1 (BSL-1)...distinguish between different types of hazards and to provide protective measures for each level, as each level...

  8. Affinity Purification of Recombinant Antibodies with Protein A or Protein G

    Type
    Protocol
    ...Update: February 7, 2022 Protocol Video Watch this instructional video to learn how to affinity purify ...3216-03 5% Sodium Azide, 500 mL, VWR 7144.8-16 PBS, 1X pH 7.4, VWR 45000-446 Benzamide, Millipore Sigma...Plasmid Collection Antibody Blog Posts Antibody Guide Transfection for Recombinant Antibodies Immunocytochemistry...reproducibility in science. Safety Warnings Sodium azide is toxic. When handling, use proper personal protective...laboratory coat, laboratory goggles, and gloves. Sodium azide containing waste should be properly disposed of ...dibasic, Millipore Sigma S7907-500G 1 M Trizma hydrochloride solution pH 9.0, Millipore Sigma T2819-1L 250... capacity. If your sample exceeds the capacity, divide the sample among multiple columns. For information...

  9. Western Blot

    Type
    Protocol
    ...General Guidelines Acrylamide percentage varies in SDS-PAGE gels. Use lower percentage acrylamide when immunoblotting...Incubate with secondary antibody Video Watch this instructional video to learn how to use western blotting...antibody, such as ideal blocking buffer and optimal antibody concentrations. Consider titrating your antibody...Plasmid Collection Antibody Blog Posts Antibody Guide Immunocytochemistry Protocol Recombinant Antibody...molecular weight proteins and higher percentage acrylamide when immunoblotting low molecular weight proteins...but will generally be between 250–1000 µL . The ideal lysis buffer will vary depending on the cellular...amount of total protein for each sample. Pro-Tip The ideal total protein loaded will vary between samples and...

  10. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ...contents of any literature. Literature are provided to you as a guide and on an “AS IS” “AS AVAILABLE” basis...Addgene 2006, All Rights Reserved. This protocol is provided for your convenience. See “warranty information...that can be accessed after a free registration. Guidelines for designing siRNAs with effective gene silencing...found, search for NAR(N 17 )YNN, where N is any nucleotide, R is a purine (A,G), and Y is a pyrimidine (...targeting introns. Avoid stretches of 4 or more nucleotide repeats, especially repeated Ts because polyT...polymerase III. Note that these were the prevailing guidelines when the pLKO.1 vector was created. Additional...program. Select sequences that have at least 3 nucleotide mismatches to all unrelated genes. TIP: Addgene...

  11. Protocol - How to Design Primers

    Type
    Protocol
    ...repeatedly the reactive groups on a nucleotide when adding a nucleotide one at a time. The main property ...more than one nucleotide at that position termed a mixed site. One can also use a nucleotide-based molecular...instead of a regular nucleotide for broader pairing capabilities. Taking into consideration the information...GCGGCG-restriction site-your sequence). Protocol Video Watch the protocol video below to learn how to design primers... Gel Electrophoresis Primer Design for PCR Oligonucleotide primers are necessary when running a PCR reaction.... They are synthesized chemically by joining nucleotides together. One must selectively block and unblock... the primer usually has stretches of several nucleotides. Also, both of the 3’ ends of the hybridized ...

  12. Using a Light Microscope Protocol

    Type
    Protocol
    .... The ocular lens also provides magnification and the power should be provided on the microscope; often...sample. Place your slide (or other sample type) on the microscope stage. If using a slide, you can secure... your focus slowly so that you don't accidentally hit your slide and risk damaging the lens or your sample...Intro to the Lab Bench Check out more protocols and videos to help you get started in the lab! Introduction...which you can control using the focus knobs on the side of the microscope. You may be wondering why there...different magnifying powers. When you look at the side of an objective, you will see several pieces of ...often this lens provides 10x magnification. To determine the final magnification of your image, multiply...

  13. Protocol - How to Perform a Diagnostic Digest

    Type
    Protocol
    ... Last Upload: May 18, 2018 Protocol Video Watch the protocol video below to learn how to analyze a restriction...Although this is never an ideal cloning strategy, sometimes it cannot be avoided. If you do have to do so...center (ideally around 1/3 of the way from one end), and also cuts in the backbone on one side of the ...recognize, restriction digests have become the most widely used method scientists employ to selectively move...including Addgene’s Sequence Analyzer , allow you to identify which restriction sites are present in a given...insert and the 5000bp backbone. Verifying Plasmid Identity by "Plasmid Fingerprinting" Often, it will be ...restriction enzymes for this approach, it is often a good idea to choose two different enzymes that will give you...

  14. Kit Free RNA Extraction

    Type
    Protocol
    ...precipitation, and resuspension. This protocol provides two options: using Solution D (see reagent section...are also included: using Isopropanol or Lithium Chloride. This protocol was adapted from (Link opens in...2006 and (Link opens in a new window) TRIzol® User Guide from ThermoFisher Scientific . Figure 1: A diagram...for precipitation step, Option A) 7.5 M Lithium Chloride (for precipitation step, Option B) Glycogen (Optional...phase without disturbing the interphase layer, consider using a lower volume pipettor like a p200 to collect...(s). You can use either Isopropanol or Lithium Chloride for this step. Isopropanol (Option A): Add 1 volume...aqueous layer. Incubate at -20°C for 1 hour. Lithium Chloride (Option B): LiCl selectively precipitates RNA ...

  15. Water Bath Protocol

    Type
    Protocol
    ...general protocol suits almost any water bath. Video Watch the video for tips on using a water bath in the lab...Intro to the Lab Bench Check out more protocols and videos to help you get started in the lab! Introduction...solution or a 70% ethanol solution to wipe down the inside of the water bath. Pro-Tip To properly maintain...maximum fill capacity is. Look for a “fill” line inside the water bath or read the user manual. If you’... level to rise up and flow over the edge of the slide. Fill the water bath with distilled water, not tap...the water bath is often better than writing on the side of the tube. This way, as items are floating, you...to necessarily maneuver the bottles or tubes to identify them. Once the water bath is at the correct temperature...

  16. Protocol - How to Run an Agarose Gel

    Type
    Protocol
    ...Last Update: Feb. 20, 2018 Protocol Video Watch the protocol video below to learn how to perform gel electrophoresis... DNA fragment by running it on an agarose gel alongside a DNA ladder (a collection of DNA fragments of...Microwave Reagents TAE ( recipe here ) Agarose Ethidum bromide (stock concentration of 10 mg/mL) Gel loading buffer...eruptive boiling can occur. Pro-Tip It is a good idea to microwave for 30-45 sec, stop and swirl, and ... flask), about 5 mins. Optional : Add ethidium bromide (EtBr) to a final concentration of approximately...be pushed away from the well comb or towards the sides/edges of the gel with a pipette tip. Place newly...Note: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and ...

  17. What is Polymerase Chain Reaction (PCR)

    Type
    Protocol
    ...size by gel electrophoresis . Protocol Video Watch the protocol video below to learn how to perform Polymerase...template), a researcher can create thousands of identical copies using a simple set of reagents and a basic...DNA and the Taq polymerase, PCR requires free nucleotides [dNTPs; adenine (A), cytosine (C), guanine (G...also requires two unique single stranded DNA oligonucleotide (oligo) primers, which anneal to the regions...and the hydrogen bonds are broken between the nucleotide base pairs. The initial denaturation step is ...have been added, pipet the forward primer onto one side of the tube wall and the reverse primer onto the...25mM MgCl 2 and/or 1µl DMSO to each reaction. The ideal setup for this troubleshooting step is to do one...

  18. Antibody Validation Using the Indirect ELISA Method

    Type
    Protocol
    ...Recombinant Antibody Purification Protocol Antibody Guide Introduction This protocol describes how to run ...support reproducibility in science. General Considerations The ELISA can be completed in a single day ...different concentrations of antigen to determine the ideal concentration. Prepare serial dilutions of the purified...remove the plate seal from the 96-well plate and set aside. Aspirate the wells and use a multichannel pipette...concentrations in the antibody dilution buffer. Pro-Tip The ideal antibody concentration will vary and needs to be...

  19. Personal Protective Equipment (PPE) for BSL-1 and BSL-2 Labs

    Type
    Protocol
    ...additional precautions that lie beyond PPE. Video Watch the video for tips on using PPE in the lab. Equipment...These guidelines summarize the proper personal protective equipment (PPE) for BSL-1 and BSL-2 labs. Protocols...Intro to the Lab Bench Check out more protocols and videos to help you get started in the lab! Introduction...There are four biosafety levels. This protocol provides information for both biosafety level 1 (BSL-1)...are meant to protect individuals from potential accidents such as spills. BSL-2 is different because it ...Gloves Eye protection and face shields, as needed Guidelines Your lab coat should be buttoned to protect your..., be aware that some gloves may contain latex. Consider nitrile gloves as an alternative. For extra precautions...

  20. Protocol - How to Perform Sequence Analysis

    Type
    Protocol
    ...primer should be a minimum of 50 nucleotides and a maximum of 300 nucleotides from your target. Are there ...than simply looking at the bases provided by your sequencing provider. My sequence has “N”s in it- what...etc.) involves selecting one or more unique oligonucleotide primers that flank the regions of your plasmid... experiments. There are a number of factors to consider when selecting sequencing primers, including: ...that can be used for sequencing? There are many widely used, common primers that are often found within...colored peaks corresponding to one of the four nucleotide bases. This is an example of a trace file from...sequencing result The original trace file (.ab1 file) provided by your sequencing company or core facility The...

  21. Lentivirus ddPCR Titration

    Type
    Protocol
    ...green) and negative (black) droplets Sample ID Plasmid ID Dilution Copies/20 µL RRE (FAM) Copies/20 µL...Titration by qPCR Addgene Viral Service Lentivirus Guide Virus Blog Posts Introduction This protocol describes...bleach. Safety Warnings Lentivirus is generally considered biosafety level 2+. Please ensure that you are...plate onto a chilled 96-well freezer block and set aside in the BSC to cool. Prepare the RRE/RPP30 master...bottom of the tube without collecting drops along the side of the tube. Add 4 µL of the 25 ng/µL samples to...bottom of the well. Lift the tips ~1mm. Touch the side of the well and tilt the pipette tips at a 45° angle...slowly and gently dispensing the droplets down the side of the tube. Place a Pierceable Foil Heat Seal on...

  22. AAV Production in HEK293 Cells

    Type
    Protocol
    ... pH 8.5 + 60 mL of 5 M Sodium Chloride + 4 mL of 1 M Magnesium Chloride Close the bottle and mix by inverting...may also like... AAV Viral Service Viral Vector Guides Virus Blog Posts Molecular Biology Reference Introduction...surface area as 21 x T-175 flasks). Cell stacks provide an efficient means to scale-up without having to...protection Vortex Reagents Adherent HEK293T cells (ideally AAV-293T clones) Pro-Tip While adherent, these ... solution and use hydrochloric acid or sodium hydroxide to adjust the pH to 7.0. Pro-Tip The pH of this... through a 0.22 μm membrane. Store at 4 °C. Considerations Before You Start The health of the HEK293T ...allow cells to reach 100% confluence (80–90% is ideal). Pass and plate the cells the day before the transfection...

  23. Pipetting Protocol

    Type
    Protocol
    ...the pipette. Last Update: September 2022 Video Watch the video for tips on pipetting in the lab. Equipment...Intro to the Lab Bench Check out more protocols and videos to help you get started in the lab! Introduction...impact your experimental results. This protocol will guide you in selecting the correct pipette and pipette... pushes the pipette tip off of the pipette. This side view photo of a P1000 pipette shows the volume adjustment...range that the tip can hold. This should give you an idea of what pipette to use with it. The purpose of the...the liquid and avoid touching the pipette to the inside of the tube. Only the tip should be in the liquid...from the container making sure not to touch the sides of the container with the pipette. Discard the pipette...

  24. Ligation Independent Cloning

    Type
    Protocol
    ...will include the free nucleotide dGTP in the reaction (exclude all other nucleotides from standard polymerase...instructions provided by your polymerase manufacturer. It is very important to remove all free nucleotides from...may also like... Cloning Methods Empty Backbone Guide Molecular Biology Protocols Overview Ligation Independent...short overlapping regions (usually 4 bp) does not provide enough stability to hold the plasmid together through...linearized vector from the reaction mixture, and may provide counter-selection for negative clones as in the...The manufacturer of an LIC-specific vector will provide the homologous sequence which must be built into...

  25. Plasmid Cloning by PCR (with Protocols)

    Type
    Protocol
    ...important to use a high fidelity taq polymerase to minimize mutations. The fidelity of the polymerase becomes...we recommend that you use a wide gel comb, run the gel on the slower side, and skip lanes between samples... Addgene’s Sequence Analyzer , to allow you to identify which restriction sites are present in a given... chance for mis-priming, so you can use a pretty wide range of annealing temperatures, but you may need...addition to a DNA ladder standard, it is also a good idea to run an uncut sample of each vector to help with...total DNA in a standard ligation reaction. You ideally want a recipient plasmid to insert ratio of approximately...

  26. Gibson Assembly Protocol

    Type
    Protocol
    ...should have identical sequences on the ends (sequences A and B in the figures). These identical sequences... each other. Phusion High-Fidelity DNA Polymerase - incorporates nucleotides to “fill in” the gaps in ...may also like... Cloning Methods Empty Backbone Guide Molecular Biology Protocols Summary In 2009 Dr. ...PCR with primers that contain a 5′ end that is identical to an adjacent segment and a 3′ end that anneals...opens in a new window) OpenWetWare: Janet Matsen’s guide to Gibson Assembly (Link opens in a new window) ...

  27. Immunocytochemistry

    Type
    Protocol
    ...the microscope slide. Gently place the coverslip on the microscope slide with the cell side facing down....Plasmid Collection Antibody Blog Posts Antibody Guide Western Blot Protocol Recombinant Antibody Purification...antibody Secondary antibody Deionized water Microscope slide Anti-fade mounting medium Laboratory wipes 15 mL...and follow your institution's laboratory safety guidelines for disposing of waste in the appropriate container...instructions before starting your experiment and consider titrating your antibody to determine the optimal...

  28. CRISPR Library Amplification

    Type
    Protocol
    ...Amplification You may also like... Pooled libraries Guide to Using Pooled Libraries All CRISPR Pooled Libraries... plasmids differ by only a short region of DNA: guide RNAs targeting different genes in an organism's ...applications. Repeated amplifications should be avoided as best as possible due to the inherent possibility... abrasively at a certain angle. Incubate plates upside down at 30 ℃ overnight. Critical Ensure at this...media drips onto the lid. Let plates remain agar side up until dried before overnight incubation if needed... number of colonies can be too great to count. Ideally, dilutions would have been sufficient to enumerate...beforehand! If you’ve already gone too far, weigh an identical empty tube. It will be close enough for the purpose...

  29. Pouring LB Agar Plates

    Type
    Protocol
    ...Protocol Video Before you begin this protocol, watch the protocol video below to get a quick idea of how...extract 10.0 g peptone from casein 10.0 g sodium chloride 12.0 g agar-agar 1 L Sterile H 2 O Sterile plates...and the medium they will contain including the identity of the antibiotic. Pro-Tip We batch label our ...particular antibiotics. Position the flame just to the side of where you’ll be pouring your plates - be sure... with a pipete for the first plate to get a good idea of what that volume looks like in your particular...

  30. AAV ddPCR Titration

    Type
    Protocol
    ...Titration by qPCR AAV Viral Service Viral Vector Guides Virus Blog Posts Molecular Biology Reference Introduction... 10% bleach. Safety Warnings AAV is generally considered biosafety level 1 but may require BSL-2 handling...plate onto a chilled 96-well freezer block and set aside in the droplet generation BSC to cool. Prepare the...bottom of the tube without collecting drops along the side of the tube. Cap gently - do not push the cap in...bottom of the well. Lift the tips ~1 mm. Touch the side of the well and tilt the pipette tips at a 45° angle...slowly and gently dispensing the droplets down the side of the tube. Place a Pierceable Foil Heat Seal on... should be close to zero (B08). At Addgene, we consider runs with an NTC > 10 to be invalid. To reduce...

  31. Molecular Biology Protocol - Restriction Digest of Plasmid DNA

    Type
    Protocol
    ...check the identity of a plasmid by diagnostic digest . Last Upload: Oct. 11th, 2016 Protocol Video Watch ...restriction enzymes, allowing scientists to target a wide variety of recognition sequences. For a list of ...Watch the video below to learn how to analyze your restriction digest results. Equipment Electrophoresis chamber...restriction enzyme activity is determined under ideal conditions with very clean DNA, so using a little...enzymes cut sequences which are similar, but not identical, to their recognition sites. This is due to "...

  32. Transfection for Recombinant Antibodies

    Type
    Protocol
    ...support reproducibility in science. General Considerations For antibody-expressing plasmids containing...antigen (EBNA). Safety Warnings HEK293 cells are considered biosafety level 2. Please ensure that you are...tubes Microcentrifuge tubes Cell counting chamber slide 30 mL luer-lock syringe, BD BD302832 0.2 µm luer-lock...salt, Sigma Aldrich P4543-10G Polyethylenimine hydrochloride, M.W. 40000 (PEI-MAX), Linear, Transfection ...BalanCD HEK293 Feed, Irvine Scientific 91166-500ML Benzamide, Millipore Sigma 12072 Antipain, Millipore Sigma...several hours. Adjust to pH 7.0 with 10 N sodium hydroxide (NaOH) or 5 N hydrochloric acid (HCl). Note: Adjust...

  33. Plasmid Cloning by Restriction Enzyme Digest (with Protocols)

    Type
    Protocol
    ...we recommend that you use a wide gel comb, run the gel on the slower side, and skip lanes between samples...including Addgene’s Sequence Analyzer , allow you to identify which restriction sites are present in a given...plasmid (if you are creating a fusion protein) Ideally, you will find two different restriction enzymes...addition to a DNA ladder standard, it is also a good idea to run an uncut sample of each plasmid to help with...total DNA in a standard ligation reaction. You ideally want a recipient plasmid to insert ratio of approximately...

  34. Coomassie Purity Stain of Recombinant Antibodies

    Type
    Protocol
    ...sample is separated by denaturing polyacrylamide gel electrophoresis alongside serial dilutions of a standard...gel Day 1 or later: Image analysis Video Watch this instructional video to learn how to determine purity...Plasmid Collection Antibody Blog Posts Antibody Guide Transfection for Recombinant Antibodies Recombinant...in the cell (e.g., failure to cleave the signal peptide) and may not be functional. This sample would fail...

  35. Protocol - How to Purify DNA from an Agarose Gel

    Type
    Protocol
    ...nice crisp bands. This can be achieved by using a wider gel comb and running the gel at a lower voltage....the UV exposure of the DNA. Therefore, it is a bad idea to use a gel imager to take a picture of the gel... gel. Notes: To protect the UV box, it is a good idea to place the gel on a glass plate if available. ...down on the UV box and trim the top, bottom and sides with the razor blade. This is especially important...voltage for a longer period of time; b) using a wider gel comb; or c) loading less DNA in the well. How...

  36. Molecular Biology Reference

    Type
    Guide
    ...helix. The order of these 4 nucleotides makes up the genetic code and provides the instructions to make ...Scientists often “sequence DNA” to identify the order of these four nucleotide bases in a particular DNA strand... protocols and videos for specific techniques and tips! Addgene Protocols Addgene Video Library...Addgene's molecular biology reference guide, including information on molecular genetics, plasmids, sequencing... the conclusions that genes encode proteins and reside on chromosomes, which are composed of DNA. These... transcribed from DNA. DNA is comprised of 4 nucleotides or bases, adenine, thymine, cytosine, and guanine...amino acids. Each amino acid is encoded for by 3 nucleotides termed a codon. As there are only 20 natural ...

  37. Fluorescence Titering Assay

    Type
    Protocol
    ...for Lentivirus You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols Viral Service...applications. Safety Warnings Lentivirus is generally considered biosafety level 2+. Please ensure that you are... in the lab by heating to 56 °C for 30 min. Considerations Before You Start The health of the target cell...anticipate that your viral stock will be higher titer, consider additional dilutions. Dilution Volume of Lentivirus...cells in each well. When calculating titer, only consider wells with less than 40% fluorescent cells. Titering...

  38. General Transfection

    Type
    Protocol
    ... Transfection You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols Viral Service...dish, Corning 430167 Hydrochloric acid Sodium hydroxide 0.22 μm polyethersulfone (PES) filter Syringes... the solution Use hydrochloric acid or sodium hydroxide to adjust the pH to 7.0. Typically, the solution...discard the tube and thaw a new working stock. Considerations Before You Start The health of the cell line...of pRosetta :µg of PEI. The 1:2 and 1:3 ratios provided high transfection efficiencies as can be seen ...

  39. Protocol - Bacterial Transformation

    Type
    Protocol
    ...Last Update: Nov. 13, 2017 Protocol Video Watch the protocol video below to learn how to isolate single...C water bath for 30-60 secs (45 secs is usually ideal, but this varies depending on the competent cells...outgrowth, for the other antibiotics it is a good idea to outgrow for at least 20-30 mins) I get very few...you need to transform large plasmids, it is a good idea to use electro-competent cells. Instead of relying...

  40. Plan Your Experiment

    Type
    Guide
    ... Science Guides CRISPR Guide Plan Your Experiment Plan Your CRISPR Experiment Jump To: Plan Your Experiment...selection marker to identify and enrich positive cells Packaging and envelope plasmids provide the necessary...see our Lentivirus Guide ) Stable, tunable expression of Cas9 and/or gRNA in a wide variety of mammalian... desired edit, ideally less than 10 bp away. In this case, it is necessary to identify the exact location... increase specificity, you can also consider using a high-fidelity Cas enzyme. In addition to off-target...the genome like never before. This section will provide a general framework to get you started using CRISPR...apply to using CRISPR in other organisms. First, consider the genetic manipulation that is necessary to ...
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