Skip to main content
Addgene

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

In Category

We narrowed to 934 results for: TIM;

Showing: 881 - 900 of 934 results

  1. Plasmids 101: Biotinylation

    Type
    Blog Post
    ...(McInnes et al., 1990). Photobiotinylation is sometimes easier and less expensive than other enzymatic...

  4. A Control for All Seasons

    Type
    Blog Post
    ..., you could repeat the tau western blot several times with the same samples and compare the results across...

  8. What is Polymerase Chain Reaction (PCR)

    Type
    Protocol
    ...molecule (single gene perhaps) to be copied multiple times by Taq Polymerase. From a single copy of DNA (the...for 1 minute at 72°C: The Taq polymerase has an optimal temperature around 70-75°C so this step enables... accurately and rapidly. Repeat steps 2-4 25-30 times. Final Extension for 5 minutes at 72°C: A final ...variable) Note: If you are doing multiple PCRs, save time by creating a “master mix,” which minimizes the ...instructions for specific instructions about extension time and temperatures. Initial Denaturation for 2 minutes...contents by gently pipetting up and down several times. Keep tube on ice. Add the forward and reverse primers... dH 2 O equal to the number of nanomoles of DNA times 10. For example, if your lyophilized DNA is 38.5nm...

  9. CRISPR Library Amplification

    Type
    Protocol
    ...2023 Workflow Timeline Day 1: Transform, recover, set up overnight growth (Estimated time 2-3 hours) Transformation...ensure that growth times are limited. Day 2: Harvest cells and purify DNA (Estimated time 3-4 hours) Cells...modifications dictated by the originating laboratory for optimal results. If you obtained the pooled library from...that will cut the shared plasmid backbone a single time and visualize that digestion on an agarose gel (...plates at least one day in advance to allow adequate time to fully gel and to dry slightly. We routinely use...Flick gently to mix. Electroporate cells (one at a time for a total of eight electroporations): Electroporator...Colonies may appear small and require extra incubation time in order to be enumerated accurately. Frequently...

  10. AAV Production in HEK293 Cells

    Type
    Protocol
    ...that longer incubation times can reduce transfection efficiency. Add the OptiMEM + DNA + PEI solution to...handle a large number of T-175 flasks. Workflow Timeline Day 0: Seed cells in CS2 Day 2: Seed cells in ...in this protocol, but their conditions must be optimized. Plasmids for transfection: pHelper; pRC (Rep-...addition of acid or base. Add only a few drops at a time. Allow them to mix and recheck the pH to prevent... Close the bottle and mix by inverting multiple times Adjust pH to ~8.5 Filter sterilize through a 0.22...The health of the HEK293T cells is critical for optimal AAV yield. Do not overgrow your cells. Pass the...Complete. Pipet back and forth vigorously multiple times to obtain a single cell suspension (no clumps of...

  11. Isolating a Monoclonal Cell Population by Limiting Dilution

    Type
    Protocol
    ... could lead to reduced transgene expression over time, as the lower expressing clones take over the polyclonal...individual cells with cloning cylinders. Workflow Timeline This protocol begins with a stable cell pool. ...theory a monoclonal line could be generated at any time. Considerations Before You Start Some cell lines...medium with increased serum concentrations, may be optimal for different cell types. Before starting this ... transgene expression. This protocol has been optimized for adherent cells but can be modified for suspension...because the buildup of waste products could be suboptimal for cell growth. This conditioned medium will...clumps into individual cells by passing several times through a serological pipet or by passing through...

  12. AAV Titration by qPCR Using SYBR Green Technology

    Type
    Protocol
    ...Update: February 13, 2019 Estimate of time required: ~3 h Workflow Timeline Plate set-up: 2 h qPCR run... very well by pipetting back and forth multiple times at each step Reagent Preparation Master Mix: Count...Keep track of the Ct value for each standard over time. They should remain within 0.5 Ct of their initial...the Ct value of the standard starts to drift, it’s time to make a new one. When developing the assay multiple... to pipet each dilution up and down at least 10 times, and use at least half of the final volume (mix ...mix well by pipetting back and forth at least 5 times. Seal plate with transparent film. Centrifuge at... U, Sing A, Ehrhardt A, Baiker A. Universal real-time PCR for the detection and quantification of adeno-associated...

  13. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ...harvesting your cells. However, you should optimize the time based on your cell line and assay: Assay Days...Designing shRNA Oligos for pLKO.1 B.1 Determine the optimal 21-mer targets in your gene B.2 Order oligos compatible... F.1 Recommended materials F.2 Determining the optimal puromycin concentration F.3 Protocol for lentiviral...Designing shRNA Oligos for pLKO.1 B.1 Determining the Optimal 21-mer Targets in your Gene Selection of suitable...plasmid to obtain the ratio that gives you the optimal viral production. d. Create a master mix of FuGENE...located in the “Appendix”. F.2. Determining the Optimal Puromycin Concentration Each cell line responds...Addgene strongly recommends that you determine the optimal puromycin concentration for your cell line before...

  14. Antibody Validation Using the Indirect ELISA Method

    Type
    Protocol
    ...area and avoid breathing in the vapors. Workflow Timeline Day 1: Antigen Coating Day 2: Blocking Day 3: ...incubation and plate read Tips and Troubleshooting The optimal concentration of primary antibody to use will vary...999.5 mL 1X PBS Cap the bottle and invert several times to mix. Carefully remove the plate seal from the...to 25 mL of PBS. Cap the tube and invert several times to mix. Using a multichannel pipette, add 200 µL...into 50 mL PBS . Cap the tube and invert several times to mix. Dilute the primary antibody and an isotype...between 1–10 µg/mL. Cap and invert the tubes several times to mix. Using a multichannel pipette, add 100 µL...dilution buffer. Cap and invert the tube several times to mix. Using a multichannel pipette, add 100 µL...

  15. Immunocytochemistry

    Type
    Protocol
    ... fixation method. The protocol may need to be optimized for different cells, target proteins, etc. Sharing...science. Last Update: January 20, 2022 Workflow Timeline Day 1: Seed cells Day 3-4: Fix and label cells...antibodies, such as antibody concentrations, incubation times, and recommended compatible reagents. Secondary ...concentration in antibody dilution buffer. Pro-Tip The optimal antibody concentration will vary but generally ...fluorescent filters. Tips and Troubleshooting The optimal fixation method will vary depending on the sample... find the best conditions for your target. The optimal antibody concentration will vary between antibodies...

  16. Western Blot

    Type
    Protocol
    ...buffer and optimal antibody concentrations. Consider titrating your antibody to determine the optimal dose....approaches. Last Update: January 24, 2022 Workflow Timeline Day 1: Prepare lysates, run SDS-PAGE, transfer... concentration in blocking buffer. Pro-Tip The optimal concentration will vary between antibodies but ...detect the bands. Tips and Troubleshooting The optimal lysis buffer will vary depending on the sample ... buffers to find the best for your target. The optimal concentration and blocking buffer will vary between...consider titrating your antibody to determine the optimal dose. To ensure that your antibody is both functioning...

  17. Transfection for Recombinant Antibodies

    Type
    Protocol
    ...regulations. Last Update: February 18, 2022 Workflow Timeline Day 1: Seed cells Day 2: Transfect cells Day 3...no more than 200 µL of NaOH or 20 µL of HCl at a time. Add deionized water to a final volume of 1 L and...resuspended before sampling. Gently swirl the flask 5–10 times before sampling. Transfer 10 µL of trypan blue into...microfuge tube containing the trypan blue. Pipette 10 times to mix. Load 10 µL of the cell suspension/trypan...the tube and vortex for 5 s to mix. Pro-Tip The optimal ratio of DNA:PEI may vary significantly and should...the flask dropwise. Cap the flasks and swirl 5–10 times to mix. Return the flask to the incubator. Section... Feed. Pro-Tip The feed can be repeated up to 4 times for a total of 16% of the culture volume. At 72-...

  18. General Transfection

    Type
    Protocol
    ...reagents. Workflow Timeline Day 0: Seed HEK293T cells (or a subclone of HEK293T optimized for viral production...this protocol with a subclone of HEK293T cells optimized for viral production (AAVpro or Lenti-X), but ...addition of acid or base. Add only a few drops at a time, allow them to mix and recheck the pH to prevent...levels of virus. HEK293T cells should be split 3 times a week: Monday: Plate 1x10 6 cells in a 75 cm 2 ...are below passage 20 for viral production. The optimal mass DNA:mass PEI ratio will need to be empirically...

  19. Protocol - Bacterial Transformation

    Type
    Protocol
    ... cells, which come frozen and are prepared for optimal transformation efficiencies upon thawing. For the...flicking the bottom of the tube with your finger a few times. Pro-Tip Transformation efficiencies will be approximately... Pro-Tip This outgrowth step allows the bacteria time to generate the antibiotic resistance proteins encoded...plates at 37°C overnight. Tips and FAQ How can I save time when carrying out transformations? If you are not...grow up more of the plasmid) you can save a lot of time by shortening or skipping many steps and will still...your transformation procedure is working. TIP: Sometimes less is more. Although it may be counter-intuitive...

  20. Protocol - How to Ligate Plasmid DNA

    Type
    Protocol
    ...ratios to optimize the ligation reaction. See Tips and FAQ below for details on optimization. Incubate...complementary, the two pieces of DNA connect and ultimately are fused by the ligation reaction. The example...is a good idea to take a fresh tube, thaw it one time and aliquot individual tubes of 5, 10 or 20μL for...plasmid in ligation or transformation reagents Optimizing the Vector:Insert Ratio Although a 3:1 insert...to vector ratio is usually sufficient, you can optimize the amount of insert and vector to improve ligation...
Showing: 881 - 900 of 934 results