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

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We narrowed to 49 results for: ple

Showing: 31 - 49 of 49 results

  1. General Transfection

    Type
    Protocol
    ...DMEM complete containing 25 µM chloroquine diphosphate and incubate ~5 h For 10 mL of DMEM complete, add... of DMEM complete. Incubate the cells 24–48 h before checking for protein expression. Sample Data Legend...Syringes for filtering Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine (or...HEK293T cells at 3.8x10 6 cells per plate in DMEM complete in 10 cm tissue culture plates. Incubate the cells...

  2. Antibody Validation Using the Indirect ELISA Method

    Type
    Protocol
    ... unknown sample (don’t forget to factor in that dilution when calculating the original sample concentration...similar outcomes when using these protocols. However, please be aware that the protocol may need to be adjusted...science. General Considerations The ELISA can be completed in a single day or broken up over several days...is a suggested starting point. If your unknown sample’s absorbance falls above the range of the standard...concentration!). Sample Data Figure 1: The plate was coated with serial dilutions of human recombinant purified...

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

    Type
    Protocol
    ...stretch of DNA to a plasmid, such as: Changing a Multiple Cloning Site (MCS) Adding short tags - ex: HA ...sites and add them to your existing vector. It is a couple days of work that will pay off for years to come...bp We also need to include additional bases to complement the overhangs generated when digesting the vector...could leave off the 3’ G on each oligo (and the complementary C of the other oligo), but this would destroy... competent bacteria and plate. Be sure to pick multiple colonies for mini-prepping and verify insert by...

  4. Protocol - How to Perform Sequence Analysis

    Type
    Protocol
    ... one of the four nucleotide bases. This is an example of a trace file from a high-quality portion of ...that these base calls are unreliable. This is an example of a trace file from a portion of a sequencing ...contact Addgene about the accuracy of your plasmid, please email [email protected] and include: The sequencing...trace file. If the mutation is not an artifact, please email [email protected] with the sequence, trace... you can see that just after base 70 there are multiple peaks in the same location. Looking at the trace...

  5. Lab Safety for Biosafety Levels One and Two

    Type
    Protocol
    ...that don’t cause disease in healthy humans, for example, non-pathogenic E . coli . BSL-2 is for labs that...your workspace with appropriate materials. For example, you may use 70% ethanol, quaternary ammonium, ...pathogens or chemicals that present a danger to people and the environment in biohazard containers. Biohazardous...containers that can be autoclaved. Conclusion Although simple, following appropriate BSL-1 and BSL-2 protocol...reuse or repurpose this SOP in another location, please note that you do so at your own risk; you should...

  6. Protocol - How to Ligate Plasmid DNA

    Type
    Protocol
    ...is physically attached to the backbone and the complete plasmid can be transformed into bacterial cells...overhanging base pairs on the vector and insert are complementary, the two pieces of DNA connect and ultimately...ultimately are fused by the ligation reaction. The example below depicts the ligation of two sticky ends that... will allow you to verify that the vector was completely digested and if phosphatase treated, that the...phosphatase treatment worked. This control should, in principle, be free of colonies, but the reality is that ...

  7. Immunocytochemistry

    Type
    Protocol
    ...similar outcomes when using these protocols. However, please be aware that the protocol may need to be adjusted... the host species of the primary antibody. For example, use an anti-mouse secondary antibody for primary...optimal fixation method will vary depending on the sample type and the protein of interest. You may need ...expected and specific, include a positive control sample that you know expresses the protein, such as cells...the protein of interest, and a negative control sample such as cells that do not express the protein of...

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

    Type
    Protocol
    ...between samples. In addition to a DNA ladder standard, it is also a good idea to run an uncut sample of each...location in your recipient plasmid (usually in the Multiple Cloning Site (MCS)), but do not cut elsewhere ...Annealed-oligo Cloning . If you are lucky enough to have multiple options for enzymes that flank your insert and...gel purification step, it is important to digest plenty of starting material. We recommend 1.5-2μg of donor...

  9. Protocol - Bacterial Transformation

    Type
    Protocol
    ...transforming with very small amounts of DNA or if you're multiple plasmids at once. To save money, many labs also...their own competent cells. This is a relatively simple procedure and is useful for performing low efficiency...so when higher efficiency is needed follow the complete protocol. Thaw the competent cells in your hand...outgrowth (for Ampicillin resistance it is ok to completely skip the outgrowth, for the other antibiotics...

  10. Pipetting Protocol

    Type
    Protocol
    ...different samples without cross contamination as long as the tip is changed between samples. Tips can ...pipette ready, push down the plunger. There will be multiple “stops” on the plunger, where you feel resistance... you may consider using a multichannel pipette. Please watch Addgene’s protocol on multichannel pipetting...

  11. Handling Plasmids from Addgene - Purifying Plasmid DNA

    Type
    Protocol
    ...of DNA Samples Add an equal volume of TE-saturated phenol-chloroform to the aqueous DNA sample. Pro-Tip...colonies and to generate liquid bacterial cultures , please see those pages. Several companies, such as (Link...remaining contaminant proteins and RNase A from the DNA sample. When phenol is mixed with the aqueous solution...

  12. Protocol - How to Create a Bacterial Glycerol Stock

    Type
    Protocol
    ...before you place the sample at -80°C. Frozen tubes are hard to write on and samples stored for long periods...streaking onto LB agar will prevent it from thawing completely and will improve the shelf life. It is very important...

  13. Protocol - Over-Agar Antibiotic Plating

    Type
    Protocol
    ...bacteria containing the plasmids of interest (for example, fewer satellite colonies will grow). It is, however...over-agar for selection of transformed E. coli . Sample Data Selection of E.coli on LB-agar using different...of Carbenicillin stock used (150 µL per plate). Please note we have found that there is generally a broad...

  16. Weighing Reagents Protocol

    Type
    Protocol
    ...you can weigh out the reagents in batches. For example, if you need 300 g of sucrose, you can weigh out...

  17. Water Bath Protocol

    Type
    Protocol
    ...water baths with instructions on the bottle, for example, number of drops per liter. Place a thermometer...
Showing: 31 - 49 of 49 results