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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 28 results for: SEC

Showing: 1 - 20 of 28 results

  1. AAV ddPCR Titration

    Type
    Protocol
    ...Vortex primers, probe and master mix for 15 sec then spin 10 sec in a mini centrifuge and place on ice. Wipe...Vortex the master mix for 15 sec and spin in a mini centrifuge for 10 sec before use. Place an 8-well ...Cycling Step Temperature (°C) Time (min) Ramp Rate (°C/sec) # Cycles Denaturation 95 10 2 1 Denaturation 95 ...cartridge with the DG8 gasket, making sure that it is secure. Transfer the cartridge holder to the droplet generator...

  2. Lentivirus ddPCR Titration

    Type
    Protocol
    ...primers/probe mixes and master mix for 15 sec then spin 10 sec in a mini centrifuge and place on ice. Wipe...Vortex the master mix for 15 sec and spin in a mini centrifuge for 10 sec before use. Place an 8-well ...Cycling Step Temperature (°C) Time (min) Ramp Rate (°C/sec) # Cycles Enzyme Activation 95 10 2 1 Denaturation...cartridge with the DG8 gasket, making sure that it is secure. Transfer the cartridge holder to the droplet generator...

  3. Protocol - Bacterial Transformation

    Type
    Protocol
    ...of the tube into a 42°C water bath for 30-60 secs (45 secs is usually ideal, but this varies depending ... you plate 50 μL on one plate and the rest on a second plate. This gives the best chance of getting single...

  4. Handling Plasmids from Addgene - Purifying Plasmid DNA

    Type
    Protocol
    ... Centrifuge in microfuge tube at 10,000 g for 30 sec. Pour off the supernatant, being careful not to disturb...is not available. Vortex microfuge tube for 30-60 sec. Centrifuge the tube for 5 min at room temperature...

  5. AAV Titration by qPCR Using SYBR Green Technology

    Type
    Protocol
    ...using SYBR detection: 98 °C 3 min / 98 °C 15 sec / 58 °C 30 sec / read plate/ repeat 39x from step 3 / melt...a single peak should be seen. The presence of a second peak at a temperature of ~70–75 °C usually indicates...

  6. AAV Production in HEK293 Cells

    Type
    Protocol
    ...ratio). Shake the bottle up/down vigorously for 30 sec (it’s okay to make bubbles). Incubate at RT for 15... all resuspended cell pellets and sonicate 5 x 1 sec pulses with at least 5 minutes on ice between each...

  7. Protocol - How to Run an Agarose Gel

    Type
    Protocol
    ...Pro-Tip It is a good idea to microwave for 30-45 sec, stop and swirl, and then continue towards a boil...the sample is unloaded, push the pipettor to the second stop and carefully raise the pipette straight out...

  8. Western Blot

    Type
    Protocol
    ... anti-mouse secondary antibody for primary antibodies raised in a mouse. Procedure Section 1: Lyse cells...incubate with primary antibody Day 2: Incubate with secondary antibody Video Watch this instructional video ...Chemiluminescence substrate Plastic wrap Primary antibody Secondary antibody Deionized water Before Starting Refer... your antibody to determine the optimal dose. Secondary antibodies must match the host species of the ...immediately or store at -80 °C until ready to use. Section 2: Determine the total protein concentration and...to 1X . Boil the samples for 10 min at 100 °C . Section 3: SDS-PAGE Prepare the precast gel as follows:...separation. Gently open the gel with the spatula. Section 4: Dry transfer Soak the gel for 15 min in 20% ...

  9. Antibody Validation Using the Indirect ELISA Method

    Type
    Protocol
    ... room temperature or overnight at 4 °C . Section 4: Secondary antibody incubation Carefully remove the...Blocking Day 3: Primary antibody incubation Day 4: Secondary antibody incubation and plate read Tips and Troubleshooting...control antibody HRP-conjugated isotype-specific secondary antibody ELISA 96-well microplate, Corning 9018...Starting Warm reagents to room temperature. Procedure Section 1: Prepare the Antigen Standard and coat the plate...incubate at 37 °C for 30 min , or overnight at 4 °C . Section 2: Block the plate Prepare the wash buffer (0.05%...2 h at room temperature or overnight at 4 °C . Section 3: Primary antibody incubation Carefully remove...wells. Dilute the HRP-conjugated isotype-specific secondary antibody to the desired concentrations in the ...

  10. Immunocytochemistry

    Type
    Protocol
    ...antibody against a target protein and a fluorescent secondary antibody....antibody against a target protein and a fluorescent secondary antibody. This protocol outlines the steps for...serum albumin (BSA) Triton X-100 Primary antibody Secondary antibody Deionized water Microscope slide Anti-fade...incubation times, and recommended compatible reagents. Secondary antibodies must match the host species of the ...primary antibody. For example, use an anti-mouse secondary antibody for primary antibodies raised in a mouse...solution into 5 mL PBS. Protect from light. Procedure Section 1: Seeding cells Place a sterile poly-D-lysine ...to grow to the desired density before labeling. Section 2: Fixing and permeabilizing cells Gently aspirate...

  11. Protocol - How to Streak a Plate

    Type
    Protocol
    ...horizontal lines in another section of the plate, and then diagonal lines in another section of the plate. Make...the first) in each new section crosses at least one line of the previous section so that it will contain...through streak #1 and spread the bacteria over a second section of the plate, to create streak #2. Using a ...the bacteria. Gently spread the bacteria over a section of the plate, as shown in the diagram above, to...streak #2 and spread the bacteria over the last section of the plate, to create streak #3. Incubate plate...

  12. Pipetting Protocol

    Type
    Protocol
    ...read differently on each type of pipette. This section will help you understand how to read each display...065. P200: The first digit is hundreds of µL, the second is tens, and the third is ones. Therefore 100 µL...as 095. P20: The first digit is tens of µL, the second is ones, and the third (red) digit is tenths. Therefore... as 022. P2: The first digit is ones of µL, the second red digit is tenths, and the third (red) digit ...decimal place. For the P20, this represents the second decimal place, and for the P2 this represents the...for an accurate measurement in the next step. The second stop is meant to expel excess liquid from the tip...slowly press down on the plunger all the way to the second stop to release the liquid. Pro-Tip Dispense until...

  13. Kit Free RNA Extraction

    Type
    Protocol
    ...provides two options: using Solution D (see reagent section for recipe) or using an all-in-one acid guanidinium..., make sure to prepare solution D (see reagent section above for the recipe). If using TRIzol®, jump down... down to the Option #2 - TRIzol® Protocol section below. Homogenize or lyse tissues or cells in Solution...49:1) and then shake vigorously by hand for 10 seconds. Incubate sample(s) for 15 minutes on ice and centrifuge...in 0.5–1 ml of 75% Ethanol and vortex for a few seconds. Centrifuge for 5 minutes at 10,000 x g at 4 °C... TRIzol® used. Shake vigorously by hand for 10 seconds. Incubate the sample(s) for 2-3 minutes on ice ...

  14. Transfection for Recombinant Antibodies

    Type
    Protocol
    ...Aliquot and freeze upright at -20 °C. Procedure Section 1: Seeding cells The day prior to transfecting,...Pro-Tip Do not use cells that are over 30 passages. Section 2: Transfection Check the cell density and viability... s to mix. Add 450 µL of 1 mg/mL PEI-MAX to the second tube of 6 mL BCD TFX (for a 1 mg/mL stock solution...times to mix. Return the flask to the incubator. Section 3: BCD Feed and valproic acid supplementation During...Thursday (168 h post-transfection): Harvest antibody. Section 4: Harvest antibody At 168 hours (1 week) post-...

  15. What is Polymerase Chain Reaction (PCR)

    Type
    Protocol
    ... 30 seconds at 94°C: Continued denaturation of double stranded DNA. Anneal primers for 30 seconds at 55...minutes at 94°C. Denature for 30 seconds at 94°C. Anneal primers for 30 seconds at 55°C (or 5°C below Tm). ...45µl volume of master mix to add to each PCR tube. Secure the tops to the PCR tubes, gently tap each tube...

  16. Pouring LB Agar Plates

    Type
    Protocol
    ...prepare your plate pouring station: Find an empty section of lab bench with a working flame. Spray down the... containing the appropriate antibiotics, and a section for active pouring. Prepare your antibiotics. Prior... know to be resistant to the antibiotic. On the second plate, streak out a strain that’s not resistant...temperature and check for growth. See our sample data section below for positive and negative test results. Sample...

  17. Coomassie Purity Stain of Recombinant Antibodies

    Type
    Protocol
    ...deionized water. Gently invert to mix. Procedure Section 1: SDS-PAGE Add 5 µL of 4X sample buffer to each...Place the gel in the electrophoresis chamber and secure it. Note: Check the manufacturer’s instructions...and bottom part of the gel where dye is visible. Section 2: Staining the Gel Place the gel in a plastic ...sample would fail Addgene QC and would not be used. Section 3: Image Analysis Use ImageJ (Link opens in a new...

  18. Water Bath Protocol

    Type
    Protocol
    ... Oftentimes, the water bath will have a spot to secure the thermometer in place. Turn the water bath on...placed inside. Water bath floats can be used to secure tubes in place and water bath weights can hold ...

  19. Gibson Assembly Protocol

    Type
    Protocol
    ...annealing to the target sequence. Avoid strong secondary structures in the homology region. Hairpins in...activity of T5 Exonuclease and furthermore reduces secondary structure of ssDNA. (Rabe & Cepko, 2020). Incubate...
Showing: 1 - 20 of 28 results