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

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We narrowed to 1,053 results for: FER

Showing: 1021 - 1040 of 1053 results

  1. Coomassie Purity Stain of Recombinant Antibodies

    Type
    Protocol
    ...NP0321BOX 20X MOPS SDS running buffer, Invitrogen NP0001 4X NuPage sample buffer, Invitrogen NP0007 10X NuPage...to total 13 µL . Prepare 1X MOPS buffer Dilute 25 mL of 20X MOPS buffer into 475 mL of deionized water....with 200 µL 1X MOPS running buffer. Fill the chamber with 1X MOPS running buffer. Make sure that the chamber...may need to be adjusted to accommodate slight differences between products. Addgene does not endorse or....125 mg/mL: 10 µL of 0.25 mg/mL with 10 µL PBS Transfer 5 µL of each standard to a microfuge tube. Add... antibody to a concentration of 0.9–1.1 mg/mL. Transfer 5 µL of recombinant antibody to a microfuge tube...Procedure Section 1: SDS-PAGE Add 5 µL of 4X sample buffer to each sample. Add 2 µL 10X reducing agent to ...

  2. Handling Plasmids from Addgene - Purifying Plasmid DNA

    Type
    Protocol
    ...This is included in the resuspension buffer of most kits. Reference Page Top Index...bacteria transformed with your plasmid Resuspension buffer Denaturing solution Renaturing solution 2 mg/mL...ethanol or isopropanol 90% ethanol 70% ethanol TE buffer 3 M Na-acetate (pH 4.8) Protocol: Generalized DNA...Grow an overnight culture of bacteria . Pro-Tip Refer to appropriate DNA prep protocol for volume of bacteria...Remove the supernatant and resuspend the bacteria in buffer. Note: This step gets all of the bacteria back ...into suspension, but within a smaller volume of buffer that is compatible with the next solution. Add ... DNA off of the column using water or a neutral buffer such as TE. You will now have plasmid DNA that ...

  3. DNA Quantification

    Type
    Protocol
    ...samples have significantly different concentrations, it is possible that the difference between them has affected...and consistent readings from DNA in a buffered solution. Reference Page Top Index...Plasmid DNA Background Information During several different stages of molecular cloning, it is important to...particular substance in that liquid. Molecules absorb different wavelengths of light to varying degrees and most...concentration readings than a DNA sample dissolved in buffer (such as TE). You will get much more accurate and...

  4. Protocol - How to Perform a Diagnostic Digest

    Type
    Protocol
    ...restriction digest buffer (see manufacturer's instructions) Gel loading dye Electrophoresis buffer Verifying Total...below we want to know how to differentiate between two clones that differ only in the orientation of the...accurately sized on a gel and also such that they are different enough in size to be easily resolved from each...approach, it is often a good idea to choose two different enzymes that will give you unique but distinct...the expected products from each orientation are different sizes. Simply run the digest products on a gel...

  5. CRISPR Library Amplification

    Type
    Protocol
    ...Molecular Biology Reference How do I process my Addgene pooled library? Introduction Please refer to our pooled... pooled plasmids differ by only a short region of DNA: guide RNAs targeting different genes in an organism's...Picogreen, or Qubit. Each different DNA measurement type can report slightly different concentrations of DNA... Pooled libraries contain tens to millions of different plasmids in a single sample. They are often used...composition of the library. Bottlenecks, fitness differences, and plasmid recombination can all impact the...

  6. Lentivirus ddPCR Titration

    Type
    Protocol
    ... pipette 10–20 times. Use a new pipette tip to transfer 200 µL of the first dilution into the next dilution...min. Resuspend cells in 500 µL DMEM complete and transfer to a microcentrifuge tube. Centrifuge for 5 min...the DG8 gasket, making sure that it is secure. Transfer the cartridge holder to the droplet generator....while slowly and gently pipetting the droplets. Transfer the droplets to a prechilled PCR plate. Pro-Tip... 98 10 2 1 Hold 4 ∞ 2 1 After PCR is complete, transfer the plate to the Droplet Reader. Open the QuantaSoft...genome: Since the cellular gene RPP30 is used as a reference to the cellular genome, it is assumed that there... cells per well is 300,000 and the virus volume refers to the total volume the cells were transduced, ...

  7. Lentivirus Production

    Type
    Protocol
    ...Lentiviral Packaging plasmid(s), Envelope plasmid, and Transfer plasmid encoding your insert of interest Reagent...and 5 mL of glutaGRO. Store at 4 °C. Pro-Tip Different brands and lots of FBS can promote or inhibit ...1.3 pmol Envelope Plasmid ( pMD2.G ) 0.72 pmol Transfer Plasmid* 1.64 pmol OptiPro SFM to total volume...concentrations and ratios should be optimized for each transfer plasmid. Pro-Tip Endotoxins can inhibit transfection...PEI is 1:3 (1000 μL total per 10 cm dish). Using transfer plasmid pHAGE TRE dCas9-KRAB (total μg of plasmid...gives the highest percentage of GFP positive cells. Refer to the table below for a possible range of ratios...individual harvests are pooled. If pooling harvests, transfer the harvested media to a polypropylene storage...

  8. Pipetting Protocol

    Type
    Protocol
    ...Tips Although there are many different types of pipettes that have different mechanisms to disperse liquids...that the same pipette can be used for measuring different samples without cross contamination as long as...performs the same function, the numbers are read differently on each type of pipette. This section will help...Procedure How to Pipette Now that you know the different parts of the pipette, how to select tips, and ...pipette tip. Liquids of various viscosity have different flow rates. The more viscous a liquid, the lower... are pipetting the same amount of liquid into different tubes or into wells of a microplate, you may consider...

  9. Ligation Independent Cloning

    Type
    Protocol
    ...from the final product (no "cloning scars"). A “stuffer” sequence allows for electrophoretic separation...template sequence. 5' and 3' primers will have different leader sequences, but operate on the same principle...at two points by the enzyme BsaI, removing the stuffer sequence and sacB counter-selection marker (see... PCR products with sterile water (instead of TE buffer) to ensure optimal salt concentrations in subsequent... minutes. Volume (μl) Reagent Final Conc. 4 10X Buffer 1X 20-30 Eluted DNA 10-50 ng/μl 1 dGTP (100mM) ...your PCR product before proceeding, as they will interfere with the exonuclease activity of T4 Pol in the...

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

    Type
    Protocol
    ...restriction enzyme buffers). If you select enzymes that can function in the same buffer, it will save you... to use, you can check out our Empty Backbone Reference . Background Subcloning by restriction digest ... a fusion protein) Ideally, you will find two different restriction enzymes for your subcloning. It is...enzymes will work in the same restriction enzyme buffer (see (Link opens in a new window) New England Biolabs...expression vector and can begin your studies. Reference Page Top Index...

  11. Gibson Assembly Protocol

    Type
    Protocol
    ...reaction mix) consists of three different enzymes within a single buffer mixture and an optional SSB protein...isothermal reaction. With the activities of three different enzymes, the product of a Gibson Assembly is a...primers to include in the Gibson reaction are different than with normal PCR primers (Gibson et al. , ... than five fragments at a time. Resources and References New England Biolabs (NEB) (Link opens in a new...

  12. General Transfection

    Type
    Protocol
    ... This approach can be adapted for different cell lines and different transfection reagents. Workflow Timeline...5 mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and lots of FBS can promote or inhibit ...gives the highest percentage of GFP positive cells. Refer to the table below for a possible range of ratios...Incubate the mixture 15–20 min at RT. Carefully transfer the transfection mix to your HEK293T cells. Add...

  13. Virus Protocol - Generating Stable Cell Lines

    Type
    Protocol
    ... 5mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and FBS lots can promote or inhibit transfection...efficiency of your transduction, you will see different degrees of cell death upon antibiotic selection...polyclonal populations of resistant cells start proliferating and the individual wells become confluent, expand... meaning that the transgene has integrated in different locations in the various cells in the culture....varying expression levels of the transgene from different cells in the population. Once the polyclonal populations...

  14. Kit Free RNA Extraction

    Type
    Protocol
    ...Purification Agarose Gel Purification Molecular Biology Reference Introduction This protocol describes how to extract...ThermoFisher Scientific . Figure 1: A diagram of the different steps in RNA extraction. Before Starting RNA is...cells. Extract RNA from the homogenized sample(s).Transfer tissue/cell lysate to a 4 mL tube. Add the following...the cold room may damage it. Using a pipettor, transfer the top, aqueous phase to a new RNAse-free tube...

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

    Type
    Protocol
    ...shRNAs to any vector (the procedure will simply differ in terms of primer design). Let's assume that your...oligos The oligos should be resuspended in annealing buffer (10mM Tris, pH7.5-8.0, 50mM NaCL, 1mM EDTA) and...total volume of 50μL - add additional annealing buffer if necessary to get to 50μL. Efficient annealing...mini-prepping and verify insert by sequencing. Reference Page Top Index...

  16. Weighing Reagents Protocol

    Type
    Protocol
    ...Introduction For many experiments, you’ll need to make buffers, media, or other solutions. A key part of this ...cross-contamination with other substances. They also help you transfer the material to your tube or container. Pro-Tip...weighed out the correct amount of your reagent, transfer it into your container. Make sure that any spills...the accurate composition of your solutions and buffers. Now that you have your reagents weighed out, you...

  17. Protocol - How to Inoculate a Bacterial Culture

    Type
    Protocol
    ...FAQ What is the difference between high copy and low copy plasmids? The copy number refers to the number... one or more antibiotic resistance genes, which confer resistance to a specific antibiotic to the bacteria...350 - 400 rpm to obtain a higher cell density. Reference Page Top Index...

  18. Video Library

    Type
    Protocol
    ... a standard plasmid transformation protocol and offer some tips and tricks to reduce transformation time...when requesting samples from Addgene Technology Transfer How to Order A step-by-step walkthrough of Addgene's...Scientist Jessica Welch to discuss her career path, differences in research culture between the US and Australia... importance of international collaboration, and offers career advice for scientists looking to expand ...

  19. Colony Formation Titering Assay

    Type
    Protocol
    ...can be used to titer lentiviral preparations that confer antibiotic resistance....can be used to titer lentivirus preparations that confer antibiotic resistance. Determining the titer of...5 mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and lots of FBS can promote or inhibit ... the incubator. Incubate for 48–72 h. Pro-Tip Different cells may be more sensitive to lentivirus or to...

  20. Using a Light Microscope Protocol

    Type
    Protocol
    ...because different components of the specimen will refract and reflect the light. These differences create... of biological research and are found in many different types of laboratories. These tools allow you to...objective performs essentially the same task but has different magnifying powers. When you look at the side of...
Showing: 1021 - 1040 of 1053 results