Skip to main content
Addgene

Enter a sequence to perform a BLAST-based search of the Addgene plasmid sequence database.

Information

In Category

We narrowed to 44 results for: tes

Showing: 1 - 40 of 44 results

  1. Handling Plasmids from Addgene - Purifying Plasmid DNA

    Type
    Protocol
    ... μL of TE. Top Protocol: Phenol-Chloroform Extraction of DNA Samples Add an equal volume of TE-saturated...Denaturing solution Renaturing solution 2 mg/mL RNase A TE or water-saturated phenol-chloroform Chloroform 100%...100% ethanol or isopropanol 90% ethanol 70% ethanol TE buffer 3 M Na-acetate (pH 4.8) Protocol: Generalized...the column using water or a neutral buffer such as TE. You will now have plasmid DNA that has been purified... Water-saturated phenol-chloroform can be used if TE-saturated is not available. Vortex microfuge tube... paper towel for 5-20 min. Resuspend dry DNA with TE (10 mM Tris-HCl pH 8, 0.1 mM EDTA). Pro-Tip DNA resuspension...min. Centrifuge the tube for 5 min at 12,000 g. Notes: Pellet contains proteins, cell fragments, salt ...

  2. Kit Free RNA Extraction

    Type
    Protocol
    ...Isoamyl alcohol (49:1) 75% Ethanol RNase-free water or TE solution RNase free tubes: microcentrifuge tubes,...residual ethanol wash and add RNase-free water or TE as soon as the entire tube is dried but while the...visible. Resuspend RNA pellet in RNase-free water or TE. Quantify and assess the quality of your RNA sample...Incubate sample(s) for 15 minutes on ice and centrifuge the sample(s) for 15 minutes at 12,000 × g at 4°C ...Incubate the sample(s) for 2-3 minutes on ice and centrifuge for 15 minutes at 12,000 × g at 4°C to separate...

  3. Pouring LB Agar Plates

    Type
    Protocol
    ...number of plates you’d like to pour. For example: Because we’d like to make 20 plates, and our plates can hold... and negative test results. Sample Data In all cases below (-) indicates that the tested strain is not... Protocols Pouring LB Agar Plates Pouring LB Agar Plates You may...Sterile H 2 O Sterile plates of your desired size - we usually use 60 mm x 15 mm plates which can hold 5-10...colonies, we recommend using larger plates. Many labs use 100 mm x 15 mm plates which can hold 10 - 15 mL of ...appropriate number of plates and stack them on your lab bench. Label the plates with the date and the ...remainder of the plates, pour directly from the bottle. Pro-Tips Be sure to swirl your plates after pouring...

  4. DNA Quantification

    Type
    Protocol
    ...readings than a DNA sample dissolved in buffer (such as TE). You will get much more accurate and consistent ...ranges from 1.80-2.00). Repeat for each sample. Notes: Keep in mind that despite the accuracy of the NanoDrop...

  5. Ligation Independent Cloning

    Type
    Protocol
    ...digestion and PCR products with sterile water (instead of TE buffer) to ensure optimal salt concentrations in ...room temperature for 30 minutes. Inactivate T4 Pol by heating to 75° for 20 minutes. Volume (μl) Reagent ... for 5 minutes at room temperature, then add 1 μl of 25 mM EDTA, followed by another 5 minutes at room...Protocols Overview Ligation Independent Cloning (LIC) obviates the need for the time-consuming ligation step ...with a single enzyme, and remove the restriction sites from the final product (no "cloning scars"). A “...reaction may be stopped by heating to 75° for 20 minutes. Step 6: Anneal and Transform Mix your treated ...

  6. CRISPR Library Amplification

    Type
    Protocol
    ... bioassay plates and remove bacteria. Use one scraper for all plates. Use two 10 mL pipettes (one for ...Antibiotic 245 mm bioassay plates (Molecular Devices, X602) Pro-Tip Pour these plates at least one day in advance...Agar + Antibiotic plates. Prewarm 12 mL recovery media at 37 °C (for at least 15 minutes). Prewarm 3X LB... Agar + Antibiotic plates at 37 °C. Prewarm 8X LB Agar + Antibiotic Bioassay plates. Prechill Micropulser...Micropulser cuvettes on ice. Thaw 4 tubes of electrocompetent cells on ice for 15-20 minutes or until completely...transformed cells on each of the eight bioassay plates (two plates per tube). Distribute evenly with a sterile... edge that can scrape plates more abrasively at a certain angle. Incubate plates upside down at 30 ℃ overnight...

  7. Plasmid Modification by Annealed Oligo Cloning (with Protocols)

    Type
    Protocol
    ...existing sites in the original vector. Designing oligos To add NdeI, PacI, AscI and MfeI sites between ...EcoRI and SalI sites of the vector, we design a top oligo with each of the additional sites in tandem ( ... to start at 95°C for 2 minutes. Then, gradually cool to 25°C over 45 minutes. Ligation Dilute 5μL of ...tutorial we will discuss how to add new restriction sites to the MCS of an empty vector. However, the following...set of oligos containing your desired restriction sites and add them to your existing vector. It is a couple...oligo), but this would destroy the EcoRI and SalI sites in the final vector. Order the following oligos ...Place tube in 90-95°C hot block and leave for 3-5 minutes. Remove the hot block from the heat source (turn...

  8. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ...be worn at all times. Use plastic pipettes in place of glass pipettes or needles. Liquid waste should be...shRNA oligos are cloned into the AgeI and EcoRI sites in place of the stuffer. The AgeI site is destroyed... 10x NEB buffer 2 35 μL ddH 2 O Incubate for 4 minutes at 95°C in a PCR machine or in a beaker of boiling...PCR machine, incubate the sample at 70°C for 10 minutes then slowly cool to room temperature over the period...following manufacturer’s protocol. Plate on LB agar plates containing 100 μg/mL ampicillin or carbenicillin...swirling or gently flicking the tube. Incubate for 5 minutes at room temperature. e. Add 80 μL of FuGENE® master...gently flicking the tube. f. Incubate for 20-30 minutes at room temperature. g. Retrieve HEK-293T cells...

  9. What is Polymerase Chain Reaction (PCR)

    Type
    Protocol
    ...DNA for 2 minutes at 72°C. Repeat steps 8-10 for 25-30 cycles. Final Extension for 5 minutes at 72°C. ...oligo primers. This molecular biology technique creates several micrograms of target DNA from just a few.... Basic PCR Program Initial Denaturation for 2 minutes at 94°C: This initiation step heats the double ...Repeat steps 2-4 25-30 times. Final Extension for 5 minutes at 72°C: A final extension to fill-in any protruding...melting temperature (Tm). Set extension step at 1-2 minutes per kilobase of product depending on whether you...time and temperatures. Initial Denaturation for 2 minutes at 94°C. Denature for 30 seconds at 94°C. Anneal...denaturation of the DNA, particularly for GC-rich templates. What does each ingredient specifically do? Template...

  10. Antibody Validation Using the Indirect ELISA Method

    Type
    Protocol
    ...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment...instead of room temperature or 37 °C. The protocol notes when there are different incubation options available...empirically determined. If possible, run an initial test with serially diluted primary antibody and determine...Equipment Spectrophotometer compatible with 96-well plates 1–10 µL single channel pipette 2–20 µL single channel...VWR 76322-150 Pipette tips, 20 µL, VWR 76322-134 Pipettes, 10 mL, VWR 89130-898 1 L polystyrene bottle, ...display a green product. A precipitated product indicates the presence of too much HRP and the need to optimize... and incubated overnight with Anti-Desmin [D7]. Plates were incubated with HRP-linked anti-Human IgG secondary...

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

    Type
    Protocol
    ...restriction sites to flank your insert : You can use PCR Based Cloning and add restriction sites to the ends...as long as it is already bounded by restriction sites that are also present in the same orientation on...Analyzer , allow you to identify which restriction sites are present in a given sequence. When selecting ...recipient plasmid as well as a specifically designed test digest later to verify that the insert was cloned...a version of your insert flanked by restriction sites compatible with the recipient plasmid's MCS. However... within your insert. Adding desired restriction sites to your recipient plasmid : You can modify the MCS...

  12. AAV Titration by qPCR Using SYBR Green Technology

    Type
    Protocol
    ...dilution is appropriate). Quality of duplicates: Exclude duplicates from analyses if there is more than...-log of the expected titer of the samples being tested. Always include a No Template Control (NTC), i....small aliquot of each standard (enough for 1 or 2 plates) and store at -20 °C. Once a standard is thawed...the assay multiple plasmids containing ITR were tested. Plasmid #59462 is one plasmid that gave reliable...consistent results. Use the recommended plasmid, or test multiple plasmids to find a suitable one. Some labs...second peak at a temperature of ~70–75 °C usually indicates the presence of primer dimers which can increase...

  13. Protocol - Over-Agar Antibiotic Plating

    Type
    Protocol
    ... fits the plate. Incubate plates at 37 ℃ for 18 hours. Day 2 Observe plates for colony formation. Shown...Antibiotic Plating You may also like... Making LB Agar Plates Bacterial Transformation Recovering Plasmid DNA...source) Incubator Reagents 6 cm diameter LB/agar plates without antibiotic High concentration (100 mg/mL... the plate at room temperature for at least 30 minutes with the lid on to give the antibiotic time to ... with smaller size than the 1 mg/mL and 2 mg/mL plates and effective selection. Selection Curve of Transformed...

  14. Video Library

    Type
    Protocol
    ...Making LB Agar Plates Create plates to culture bacteria in the lab Pouring LB Agar Plates Protocol Streaking...Bacteria on Plates Isolate single bacterial colonies on an agar plate Streaking Bacteria on Plates Protocol...CRISPR Protocol Over-Agar Antibiotic Plating Create plates to culture bacteria in the lab Over-Agar Antibiotic...

  15. Pipetting Protocol

    Type
    Protocol
    ...video for tips on pipetting in the lab. Equipment Pipettes Pipette tips Waste container Containers to hold...Tips Although there are many different types of pipettes that have different mechanisms to disperse liquids...liquids, this protocol is focused on single channel pipettes. These come in standard sizes: P2, P10, P20, P200...would read as 050. Each volume display on these pipettes will also have small tick marks at the bottom....Liquids of various viscosity have different flow rates. The more viscous a liquid, the lower the flow rate...’s important for scientists to calibrate their pipettes to make sure that they are properly tuned to dispense...

  16. Coomassie Purity Stain of Recombinant Antibodies

    Type
    Protocol
    ..., VWR 76322-154 Pipettes, 5 mL, VWR 89130-896 Pipettes, 10 mL, VWR 89130-898 Pipettes, 25 mL, VWR 89130...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment...89130-900 Pipettes, 50 mL, VWR 89130-902 Gel loading tips, Corning 4853 Microcentrifuge tubes, Neptune 3745...mobility of the bands (sample AR0016 in Figure 1) indicates that the samples may not have been processed correctly...

  17. Protocol - How to Perform a Diagnostic Digest

    Type
    Protocol
    ...Given the variety of these enzymes and the unique sites they recognize, restriction digests have become ...cleave DNA at specific sequences called restrictions sites. Often, the size of the plasmid insert and vector...Analyzer , allow you to identify which restriction sites are present in a given sequence. For a list of the...either PCR - or restriction enzyme -based cloning to test individual clones before use of more expensive forms... restriction enzymes common to multiple cloning sites, will result in similar sized bands, thus making...

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

    Type
    Protocol
    ...new window) Link to Video Making LB Agar Plates Create plates to culture bacteria in the lab Watch the...molecular biology, plasmid cloning, and titering and testing your viral preparations. Addgene...your plasmids. Virus Protocols for titering and testing your virus preparations. Antibodies Protocols for...and ligation Cloning by PCR Generate restriction sites by PCR Modification by Annealed Oligo Cloning Add...

  19. Plasmid Cloning by PCR (with Protocols)

    Type
    Protocol
    ... of DNA and at the same time adding restriction sites to the ends of that piece of DNA so that it can ... cartoon, in which we are adding EcoRI and NotI sites to Your Gene of Interest (YGOI) for ligation into...amplified (usually 18-21bp). When selecting restriction sites, you should use a DNA analysis tool, such as Addgene...Analyzer , to allow you to identify which restriction sites are present in a given sequence. You want to choose...based cloning. DNA replication by PCR has error rates that range from roughly 1 per 500bp to roughly 1...

  20. Protocol - Bacterial Transformation

    Type
    Protocol
    ... on ice (approximately 20-30 mins). Remove agar plates (containing the appropriate antibiotic ) from storage...that there isn't too much liquid media on the agar plates. If the agar plate doesn't dry adequately before...the liquid and won't grow in colonies. Incubate plates at 37°C overnight. Tips and FAQ How can I save ...access to an electroporator and the appropriate cuvettes. Follow the manufacturer's instructions for each...

  21. Lentivirus Production

    Type
    Protocol
    ...lots of FBS can promote or inhibit transfection. Test a variety of brands and lots of FBS to find one ... plate in DMEM Complete in 10 cm tissue culture plates. Incubate the cells at 37 °C, 5% CO 2 for ~20 h...the table below for a possible range of ratios to test: Ratio of DNA:PEI μg of DNA μL of 1 mg/mL PEI 1:...Centrifuge the viral supernatant at 2100 rcf for 5 minutes to pellet any packaging cells that were collected...

  22. Transfection for Recombinant Antibodies

    Type
    Protocol
    ...Biosafety Cabinet 4 °C Refrigerator Micropipettes and tips Pipettor and pipettes Benchtop centrifuge compatible...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment...Reagent Preparation instructions. Wipe down all pipettes and equipment with 10% bleach prior to use. Reagent...

  23. Virus Protocol - Generating Stable Cell Lines

    Type
    Protocol
    ...transiently-transfected cells, increases reproducibility, as it eliminates the variation associated with repeated transient...and FBS lots can promote or inhibit transfection. Test a variety of brands and FBS lot numbers to find ... expanded, prepare cell stocks and/or harvest to test for protein expression. Typically, cells transduced...levels of the transgene may have reduced growth rates, especially if that transgene is toxic. Eventually...

  24. AAV ddPCR Titration

    Type
    Protocol
    ...primers, and probe on ice before use. Wipe down all pipettes and surfaces with 10% bleach. Safety Warnings ...PCR tubes, Axygen, PCR-02-FCP-C ddPCR 96-well PCR plates, Bio-Rad, 12001925 48-well dilution plate, Bio-... 1X PCR buffer (1:25,600,000) Use multichannel pipettes for the dilution series. For dilutions 1–5, use...reduce NTC values, we recommend wiping down all pipettes and equipment with 10% bleach prior to use and...

  25. AAV Production in HEK293 Cells

    Type
    Protocol
    ...lots of FBS can promote or inhibit transfection. Test a variety of brands and lots of FBS to find one ...inactivated in the lab by heating to 56 °C for 30 minutes. 0.45 μm polyethersulfone (PES) filter system, ...and add 35 mL of 0.05% Trypsin/EDTA. Wait ~2-3 minutes for cells to detach. Gently tap the sides of the... and sonicate 5 x 1 sec pulses with at least 5 minutes on ice between each pulse, 50% amplitude. Return...

  26. Lentivirus ddPCR Titration

    Type
    Protocol
    ...mixes and samples on ice before use. Wipe down all pipettes and surfaces with 10% bleach. Safety Warnings ...PCR tubes, Axygen, PCR-02-FCP-C ddPCR 96-well PCR plates, Bio-Rad, 12001925 Pierceable foil heat seal, Bio-Rad...Cell Harvest Gently aspirate media from the 6-well plates. Be sure to use a new aspirating pipette for each...and Troubleshooting We recommend wiping down all pipettes and equipment with 10% bleach prior to use and...

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

    Type
    Protocol
    ...with standard agarose gel electrophoresis, which separates DNA by their length in base pairs. Following electrophoresis...Electrophoresis Protocol with the following amendments: Notes: Gel purification is most efficient with lower %...blade, slice the desired DNA fragment from the gel. Notes: To protect the UV box, it is a good idea to place...

  28. Protocol - How to Inoculate a Bacterial Culture

    Type
    Protocol
    ...commonly used to culture bacteria in the lab. LB agar plates are frequently used to isolate individual (clonal...characterized by a cloudy haze in the media (see right). Notes: Some protocols require bacteria to be in the log...on your plasmid. If the bacteria on your LB agar plates are not fresh, you should streak your bacteria ...

  30. General Transfection

    Type
    Protocol
    ...lots of FBS can promote or inhibit transfection. Test a variety of brands and lots of FBS to find one ... plate in DMEM complete in 10 cm tissue culture plates. Incubate the cells at 37 °C, 5% CO 2 for ~20 h...the table below for a possible range of ratios to test: Ratio of DNA:PEI Amount of DNA (μg) Volume of 1...

  31. Gibson Assembly Protocol

    Type
    Protocol
    ...Gibson Cloning? No need for specific restriction sites. Join almost any two fragments regardless of sequence...unique function for the reaction: T5 Exonuclease - creates single-strand DNA 3’ overhangs by chewing back ...other. Phusion High-Fidelity DNA Polymerase - incorporates nucleotides to “fill in” the gaps in the annealed...

  32. AAV Purification by Iodixanol Gradient Ultracentrifugation

    Type
    Protocol
    ...ultracentrifugation. The arrow indicates the 60–40% interface. The vertical black line indicates the location of the...fractions once you have a good idea when the virus elutes from the gradients. In a biosafety cabinet, carefully...

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

    Type
    Protocol
    ...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment...tubes, VWR 76289-498 Before Starting Wipe down all pipettes and equipment with 10% bleach prior to use. Warm...Spectrophotometer using the A280 IgG setting. Combine all eluates with measurable protein. Determine the protein ...

  34. Western Blot

    Type
    Protocol
    ...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment...January 24, 2022 Workflow Timeline Day 1: Prepare lysates, run SDS-PAGE, transfer, block, incubate with primary...channel pipette Pipette controller Pipette tips and pipettes Spectrophotometer Heat block Mini gel tank chamber...

  35. Water Bath Protocol

    Type
    Protocol
    ...on the surfaces of the water bath as the water evaporates from the tub. Disinfectant may be added to prevent...heat up quicker. Pro-Tip Set up the water bath 30 minutes to 1 hour before you need to use it to allow the...

  36. Protocol - How to Ligate Plasmid DNA

    Type
    Protocol
    ... 0.5-1μL T4 DNA Ligase H 2 O to a total of 10μL Notes: If the DNA concentrations are low such that you...troubleshooting failed ligations. The following table indicates the various controls: Control Ligase Interpretation...

  37. Immunocytochemistry

    Type
    Protocol
    ...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment... Equipment Pipette controller Pipette tips and pipettes Rocking platform Tweezers Fluorescent microscope...

  38. Colony Formation Titering Assay

    Type
    Protocol
    ...lots of FBS can promote or inhibit transfection. Test a variety of brands and lots of FBS to find one ...solution through a 0.22 μm filter to remove any precipitates. Stain each well with 1 mL of 0.1% crystal violet...

  39. Protocol - How to Design Primers

    Type
    Protocol
    ...also needs to avoid primer-primer annealing which creates primer dimers and disrupts the amplification process...
Showing: 1 - 40 of 44 results