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We narrowed to 956 results for: REP

Showing: 221 - 240 of 956 results

  1. Antibody Validation Using the Indirect ELISA Method

    Type
    Protocol
    ...the full details of our protocols supports reproducibility and accelerates science. Here, we list the ...solely for transparency intended to support reproducibility in science. General Considerations The ELISA...reagents to room temperature. Procedure Section 1: Prepare the Antigen Standard and coat the plate Dilute ... antigen to determine the ideal concentration. Prepare serial dilutions of the purified antigen as follows...overnight at 4 °C . Section 2: Block the plate Prepare the wash buffer (0.05% Tween-20 in PBS) as follows...microplate shaker set at 400 rpm and shake for 1 min . Repeat steps 8–10 twice for a total of three washes. After...wash, aspirate the wash buffer from the wells. Prepare the blocking buffer (1% BSA in PBS) as follows:...

  2. Lentivirus ddPCR Titration

    Type
    Protocol
    ... virus on ice. Prepare the following dilution series of virus in DMEM complete: Prepare 7 microcentrifuge..., where TU is transducing units and generally represents the number of infectious viral particles. Users...Assay: RPP30, Human, Bio-Rad, 10031244 Reagent Preparation DMEM Complete: 500 mL DMEM, high glucose, pyruvate...µL of the first dilution into the next dilution. Repeat for all dilutions as indicated in the chart below...untransduced control. Pro-Tip For even seeding, prepare a batch for 10 wells with 3,000,000 cells in 13.5...concentration of each sample on a spectrophotometer. Prepare 25 ng/µL stocks of each sample. Samples can be ...immediately or stored at -20 °C until ready to use. Preparing for ddPCR Thaw samples, primers/probe mixes, and...

  3. AAV Titration by qPCR Using SYBR Green Technology

    Type
    Protocol
    ...each step Reagent Preparation Master Mix: Count the number of samples (n) and prepare master mix for an... number of genome-containing particles in an AAV prep using qPCR, SYBR Green Technology, and Addgene’s... number of genome-containing particles of an AAV prep using SYBR green technology. This protocol was validated...vector cores. Our titers were similar to those reported by these institutions. This protocol is for a ...μL Nuclease Free Water 4.7 μL 470 μL Procedure Prepare a plasmid stock of 2 x 10 9 molecules/μL to generate...plasmids to find a suitable one. Some labs have reported better results when the plasmid is linearized....min / 98 °C 15 sec / 58 °C 30 sec / read plate/ repeat 39x from step 3 / melt curve Example of plate set-up...

  4. Affinity Purification of Recombinant Antibodies with Protein A or Protein G

    Type
    Protocol
    ...the full details of our protocols supports reproducibility and accelerates science. Here, we list the ...solely for transparency intended to support reproducibility in science. Safety Warnings Sodium azide is...to room temperature (RT) before use. Reagent Preparation 1 M sodium phosphate monobasic monohydrate (NaH...column. Collect the flow through in a 250 mL bottle. Repeat steps 9–10 such that the supernatant passes through...Centrifuge at 1000 x g for 2 min to remove PBS. Repeat Steps 30–31 3x , discarding buffer from the collection...Discard the flow through into a waste container. Repeat steps 29-31 at least 4 additional times to ensure.... If the sample concentration is still too low, repeat the centrifugation until the volume of the sample...

  5. Lentivirus Production

    Type
    Protocol
    ... 2 (am): 18 h post-transfection. Remove media, replace with fresh media. Day 3-4 (am): Harvest virus Equipment...plasmid encoding your insert of interest Reagent Preparation DMEM Complete (also known as D10): 10% v/v FBS...T75 flask in 15 mL DMEM Complete. Do not add pen-strep to the media. Use cells that are below passage 15...Incubate the cells at 37 °C, 5% CO 2 for ~20 h. Prepare a mixture of the three transfection plasmids into...empirically determined. Once a batch of PEI is prepared, transfect cells with a fluorescent plasmid using...following morning, carefully aspirate the media. Replace the media with 10 mL of DMEM Complete or OptiPro...

  6. Handling Plasmids from Addgene - Purifying Plasmid DNA

    Type
    Protocol
    ... Protocol: Kit-free Alkaline Lysis Plasmid Miniprep Prepare the following solutions: Solution I - Resuspension...you can find a protocol for kit-free plasmid mini-prep at the bottom of this page. Last Update: Feb. 8,...culture of bacteria . Pro-Tip Refer to appropriate DNA prep protocol for volume of bacteria to grow (low copy...pellet the bacteria before proceeding with DNA preparation. Pro-Tip If your entire overnight culture cannot... chloroform to the recovered aqueous DNA layer. Repeat steps 2-4. Note: Phenol-chloroform is a hazardous...

  7. Pouring LB Agar Plates

    Type
    Protocol
    ...Use this protocol to prepare LB agar plates with antibiotic in your lab....samples are sterilizing in the autoclave, you should prepare your plate pouring station: Find an empty section...antibiotics, and a section for active pouring. Prepare your antibiotics. Prior to adding your antibiotic...1000x stock solution. For example: If you’ll be preparing plates with a final concentration of 100 ug/mL..., dissolve by vortexing, and filter sterilize. Prepare a water bath at 60 ℃ with sufficient water to submerge...Only the Resistant Strain Grows If everything was prepared properly, you should see growth of only the resistant...

  8. Isolating a Monoclonal Cell Population by Limiting Dilution

    Type
    Protocol
    ...conditioned medium prepared above to make a new cell solution at a concentration of 5 cells/mL. Prepare approximately...culture dish, Corning 430167 (optional) Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-...well plate. Allow the cells to grow overnight. Replace the medium on the cells and collect the used (conditioned...a hemocytometer or other cell counter. Pro-Tip Prepare a relatively dilute cell solution (<10 6 cells/.... If you choose not to use conditioned medium, prepare this cell solution in fresh complete medium. This...

  9. General Transfection

    Type
    Protocol
    ...2 (am): 18 h post transfection - Remove media, replace with fresh media Day 3 or more (am): Observe fluorescence... (PES) filter Syringes for filtering Reagent Preparation DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-...empirically determined for each new batch of 1 mg/mL PEI prepared. There may be variation between batches of PEI...empirically determined. Once a batch of PEI is prepared, transfect cells with a fluorescent plasmid using...following morning, carefully aspirate the media. Replace the media with 15 mL of DMEM complete. Incubate...

  10. Immunocytochemistry

    Type
    Protocol
    ...the full details of our protocols supports reproducibility and accelerates science. Here, we list the ...solely for transparency intended to support reproducibility in science. Last Update: January 20, 2022 Workflow...primary antibodies raised in a mouse. Reagent Preparation Permeabilization buffer: Dilute 20 µL of Triton...100 in 50 mL PBS. 300 nM DAPI working solution: Prepare a 300 µM DAPI stock solution by diluting 2.1 µL...DAPI solution to 100 µL PBS. Protect from light. Prepare a 300 nM DAPI working solution by diluting 5 µL...

  11. Protocol - How to Create a Bacterial Glycerol Stock

    Type
    Protocol
    ...15-25% glycerol. You can prepare the glycerol stock the same time you prepare your plasmid DNA. In the...able to start an overnight culture for plasmid DNA prep the following day. Tips and FAQ The optimal concentration...glycerol stock before you begin your plasmid mini-prep. Try not to freeze/thaw your glycerol stock too ...

  12. Protocol - Over-Agar Antibiotic Plating

    Type
    Protocol
    ...result represents a single experiment. For publishable data, the experiment would need to be repeated to ...their resistance genes, as one does not need to prepare separate batches of antibiotic-containing agar....or other antibiotic resistance) Procedure Day 1 Prepare carbenicillin to a concentration of 1 mg/mL – 4...

  13. Pipetting Protocol

    Type
    Protocol
    ...bottom. For the P1000, this represents the ones. For the P200, this represents the first decimal place. ...For the P20, this represents the second decimal place, and for the P2 this represents the third decimal...

  14. Protocol - How to Inoculate a Bacterial Culture

    Type
    Protocol
    ... do a mini-prep you will usually want to start 2 mL in a falcon tube, but for larger preps you might want...inoculate bacteria in liquid culture. Protocol Prepare liquid LB. For example, to make 400 mL of LB, weigh...

  15. Ligation Independent Cloning

    Type
    Protocol
    ... but nicked vector product is then repaired during the replication cycle. Empty vectors for LIC typically... plasmid together through the transformation/replication process. LIC employs long overhangs to form a...

  16. Video Library

    Type
    Protocol
    ...Purification Purify adeno-associated virus from a preparation produced in mammalian cells AAV Purification ... Inoculating a Liquid Bacterial Culture How to prepare and grow bacteria in a liquid medium Inoculating...physical titer of your adeno-associated virus preparation. AAV Titration by qPCR Using SYBR Green Technology...

  17. Protocol - Bacterial Transformation

    Type
    Protocol
    ...bacteria are used as the means for both storing and replicating plasmids. Because of this, nearly all plasmids...expression) carry both a bacterial origin of replication and an antibiotic resistance gene for use as ...sell competent cells, which come frozen and are prepared for optimal transformation efficiencies upon thawing...

  18. What is Polymerase Chain Reaction (PCR)

    Type
    Protocol
    ...the new target DNA strand accurately and rapidly. Repeat steps 2-4 25-30 times. Final Extension for 5 minutes...°C below Tm). Extend DNA for 2 minutes at 72°C. Repeat steps 8-10 for 25-30 cycles. Final Extension for...and concentration of PCR product. Master Mix Preparation Multiply the volume of each reagent by the number...

  19. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    ...table, reprogramming efficiencies ranking as low, medium or high is based on the reprogramming efficiencies... be used to package reprogramming factors. An important milestone of reprogramming is the silencing of...incomplete silencing of reprogramming transgenes, which leads to incomplete reprogramming. Additionally, lingering...rates of reprogramming when compared to MMLV-derived retroviruses, it is not as well repressed following...by terminal repeat sequences, which the PB transposase recognizes. Following reprogramming, transgenes...risk of lingering expression of reprogramming factors. Non-replicating Plasmids: Generating iPSCs with...RNA to reprogram cells is that it’s simple and efficient. It has a high efficiency of reprogramming when...

  20. Plasmids 101: Plasmid Incompatibility

    Type
    Blog Post
    ...Origin of Replication? See our Plasmids 101: Origin of Replication blog).  The replication problem Plasmids...directly inhibit replication by binding to the origin of replication and blocking replication machinery (del...inhibit plasmid replication.   Figure 1: The replication problem. Iterons are repeated sequences within...increases, RepA transcription is repressed and replication is downregulated. In the second model, termed...cell have the same replicon and/or partitioning system. Let’s start with the replicon- the part of the ...plasmid that contains the origin of replication and the replication control machinery (Need a refresher... with the same replicon are incompatible because they compete for the same replication control machinery...
Showing: 221 - 240 of 956 results