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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 47 results for: EED

Showing: 21 - 40 of 47 results

  1. Protocol - Bacterial Transformation

    Type
    Protocol
    ... as when you have a tube of plasmid DNA and just need to transform bacteria so that you can grow up more...the transformation, so when higher efficiency is needed follow the complete protocol. Thaw the competent...less efficient at taking up larger plasmids. If you need to transform large plasmids, it is a good idea to...induce membrane permeability. To do this you will need to have access to an electroporator and the appropriate...

  2. Protocol - How to Run an Agarose Gel

    Type
    Protocol
    ...concentrations of 0.7% to 2% depending on the size of bands needed to be separated - see FAQs below . Simply adjust...add EtBr to the gel and running buffer, you will need to soak the gel in EtBr solution and then rinse ... procedures, such as molecular cloning, you will need to purify the DNA away from the agarose gel. For...want to load on a gel, make 10% more volume than needed because several microliters can be lost in pipetting...

  3. Virus Protocol - Generating Stable Cell Lines

    Type
    Protocol
    ...alternative cell lines. Workflow Timeline Day 0: Seed and transduce Cells Day 2–3 (am): Remove media, ...containing selection reagent Day 3–14: Change media as needed Day 14–18: Expand and harvest stable cell lines...control well). Perform a "reverse transduction" by seeding 50,000 cells into each well of the 6-well dish....media to make the cell solution in this step. To seed the cells: Prepare a batch of cells as follows: ...

  4. Western Blot

    Type
    Protocol
    ...precast SDS-PAGE gel, and immunoblotting. Sharing speeds science. We believe that sharing the full details.... However, please be aware that the protocol may need to be adjusted to accommodate slight differences...protein is in low abundance in the sample you will need to load a greater amount of total protein. Prepare...cellular location of the protein of interest. You may need to try a variety of lysis buffers to find the best...

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

    Type
    Protocol
    ...healthy humans. BSL-2 includes all of the precautions needed in BSL-1, however there are additional precautions...shoes Gloves Eye protection and face shields, as needed Guidelines Your lab coat should be buttoned to ...such as goggles and/or face shields can be used as needed. For BSL-2 work always wear glasses/goggles in ...

  6. Protocol - How to Streak a Plate

    Type
    Protocol
    ...a glycerol stock or stab culture of bacteria and need to purify plasmid DNA from it, you will want to ...hours) at 37 °C. Pro-Tip Some plasmids or bacteria need to be grown at 30 °C instead of 37 °C. This is ...colonies. Once you have single colonies, you can proceed to Recovering Plasmid DNA or use the individual...

  7. Ligation Independent Cloning

    Type
    Protocol
    ... Ligation Independent Cloning (LIC) obviates the need for the time-consuming ligation step of traditional...protocol ). After the digest is complete, you will need to separate the linearized vector from the reaction...free nucleotides from your PCR product before proceeding, as they will interfere with the exonuclease ...

  8. Fluorescence Titering Assay

    Type
    Protocol
    ... biosafety regulations. Workflow Timeline Day 0: Seed 293T cells Day 1: Transduce cells Day 2 (am): Remove...supernatants to multiple freeze-thaw cycles. Procedure Seed 75,000 cells into each well of a 6-well dish. Prepare...integration event per cell. When the percentage exceeds 40% you risk counting cells with multiple integration...

  9. Lab Safety for Biosafety Levels One and Two

    Type
    Protocol
    ...broken glass or needles. Sharps containers must be thick walled, impenetrable by a needle, and must be able...cholerae . BSL-2 includes all of the precautions needed in BSL-1, along with additional precautions to ...

  10. Protocol - How to Ligate Plasmid DNA

    Type
    Protocol
    ...5, 10 or 20μL for storage at -20°C. Whenever you need to set up ligations in the future you can thaw a...ligation can be improved by incubation at 37°C. Proceed with bacterial transformation . Tips and FAQ Do...of DNA no matter how long it is, and therefore we need to adjust the amount of DNA used in a ligation based...

  11. Gibson Assembly Protocol

    Type
    Protocol
    ... this method extensively. Why Gibson Cloning? No need for specific restriction sites. Join almost any ... plasmid, think about what DNA segments you will need to join to create your final plasmid. Adjacent segments...Stitching” fragments together using oligos When you need intervening sequence between two PCR products, one...

  12. Using a Light Microscope Protocol

    Type
    Protocol
    ...that can fit on a microscope stage) Reagents None needed Procedure Set up your microscope by removing any...of view is centered on a region of interest. If needed, you can also use the fine focus knob (the smaller...objective! Once on the higher objective, you will likely need to re-adjust lighting and focus. Increase the power...

  13. Handling Plasmids from Addgene - Purifying Plasmid DNA

    Type
    Protocol
    ...Centrifuge the culture to pellet the bacteria before proceeding with DNA preparation. Pro-Tip If your entire ...DNA to precipitate. Centrifuge solution at high speed (at least 12,000 rpm) for 15-30 min at 4°C. Notes...from the DNA pellet. Centrifuge solution at high speed (at least 12,000 rpm) for 5 min at room temp. Pour...

  14. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ... GGC TGT TAG AGA GAT AAT TGG A 3’). TIP: You may need to adjust the sequencing conditions if the DNA polymerase...scaled to produce different amounts of virus as needed. Day 1: a. For each plasmid to be transfected, .... Change to fresh puromycin-containing media as needed every few days. h. Assay infected cells. The following...plastic pipettes in place of glass pipettes or needles. Liquid waste should be decontaminated with at ...

  15. Coomassie Purity Stain of Recombinant Antibodies

    Type
    Protocol
    ...protein content of the sample is determined. Sharing speeds science. We believe that sharing the full details... However, please be aware that your protocol may need to be adjusted to accommodate slight differences...CSV file. Select import. Choose file. Format as needed. Select the Insert tab, then Chart . This will ...

  16. Protocol - How to Create a Bacterial Glycerol Stock

    Type
    Protocol
    ...in your desired bacterial strain and you will not need to obtain more competent cells and retransform. ...want to store bacteria for a longer time, you will need to establish glycerol stocks. The addition of glycerol...

  17. Protocol - Over-Agar Antibiotic Plating

    Type
    Protocol
    ...differing in their resistance genes, as one does not need to prepare separate batches of antibiotic-containing...experiment. For publishable data, the experiment would need to be repeated to account for variability....

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

    Type
    Protocol
    ...concentration of the DNA that you isolated before proceeding to your next intended step with the now gel purified...want to load on a gel, make 10% more volume than needed because several microliters can be lost in pipetting...

  20. Protocol - How to Perform a Diagnostic Digest

    Type
    Protocol
    ...Before beginning your diagnostic digest, you will need to select a restriction enzyme or enzymes that cut... vector by a single restriction enzyme, you will need to verify that it has be cloned in the correct orientation...
Showing: 21 - 40 of 47 results