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We narrowed to 40 results for: des.1

Showing: 21 - 30 of 40 results

  1. Protocols for Molecular Biology, Plasmid Cloning, and Viral Preps

    Type
    Protocol
    ...(PPE) for BSL-1 and BSL-2 Labs Learn how to best protect yourself when working in BSL-1 and BSL-2 labs...Levels One and Two (BSL-1 and BSL-2) Safety measures for laboratories operating at BSL-1 and BSL-2 Watch the... T4 polymerase pLKO.1 - TRC Cloning Vector Cloning protocols for using the pLKO.1 vector, a backbone used...gel Watch the Video! How to Design a Primer Key considerations when designing primers Watch the Video! Sequence...Intro to the Lab Bench Introductory techniques designed to help you get started in the lab. Basic Molecular...antibody applications. Intro to the Lab Bench Name Description (Link opens in a new window) Link to Video Personal...materials on a balance Basic Molecular Biology Name Description (Link opens in a new window) Link to Video Making...

  2. Western Blot

    Type
    Protocol
    ...Prepare the chemiluminescence substrate by mixing 1:1 reagent A to reagent B. Gently incubate the membrane...Last Update: January 24, 2022 Workflow Timeline Day 1: Prepare lysates, run SDS-PAGE, transfer, block, incubate...primary antibodies raised in a mouse. Procedure Section 1: Lyse cells Centrifuge 5 x 10 6 cells for 5 min at...Gently remove supernatant. Resuspend cell pellet in 1 mL of 1X PBS and transfer to a microcentrifuge tube...preferred method for protein determination. Prepare a 50:1 Reagent A to Reagent B dilution of the BCA assay. ...side up. Block the membrane in blocking buffer for 1 h at RT on a shaking platform. Wash the membrane 3x... vary between antibodies but is typically between 1–10 μg/mL. Incubate the membrane overnight in primary...

  3. AAV Purification by Iodixanol Gradient Ultracentrifugation

    Type
    Protocol
    ...Preparation 1 M NaCl/PBS-MK buffer Dissolve 5.84 g of NaCl, 26.3 mg of MgCl 2 and 14.91 mg of KCl in 1× PBS ...gradient for AAV purification. Workflow Timeline Day 1: Purify Day 2: Buffer exchange and concentration Note...Dissolve 26.3 mg of MgCl 2 , and 14.91 mg of KCl in 1× PBS in a final volume of 100 mL. Sterilize by passing... step: mix 4.5 mL of 60% iodixanol and 13.5 mL of 1 M NaCl/PBS-MK buffer 25% iodixanol step: mix 5 mL ...Hamilton needle, taking care to avoid bubbles (Figure 1). 8 mL of 15% iodixanol step 5 mL of 25% iodixanol...disturb the gradient!** Collect Fractions Option #1 Prepare a row of roughly 20 open 1.5 mL microcentrifuge...with a 16 ga needle and start collecting 0.5 mL to 1 mL fractions per tube. Avoid the proteinaceous material...

  4. What is Polymerase Chain Reaction (PCR)

    Type
    Protocol
    ...annealing temperature step-wise by 1-2°C. The rate of DNA synthesis is ~1-2 kb/min. The extension time can...now bind to the primer DNA sequence. Extend DNA for 1 minute at 72°C: The Taq polymerase has an optimal ...DNA (10 ng-500 ng) 5 μl 10X Taq buffer with MgCl 2 1 μl dNTP mix (10 mM each nt) 2.5 μL Forward Primer ...primer melting temperature (Tm). Set extension step at 1-2 minutes per kilobase of product depending on whether...working concentration of each primer (10uM) by making a 1:10 dilution of the stock. For example, add 100µl of...Gel Procedure Primer Design and PCR Design Primers. See our protocol on how to design primers . Note: (Link...

  5. Coomassie Purity Stain of Recombinant Antibodies

    Type
    Protocol
    ...June 14, 2023 Workflow Timeline Day 1: Run SDS-PAGE and stain gel Day 1 or later: Image analysis Video Watch...lane Example for AR0018 (lane 2 in Figure 1): Sample Peak 1 (contaminant) Peak 2 (contaminant) Peak 3 ...antibodies using Coomassie stain. Equipment Heat block 1–10 µL single channel pipette 2–20 µL single channel...deionized water. Gently invert to mix. Procedure Section 1: SDS-PAGE Add 5 µL of 4X sample buffer to each sample...attach to a power supply. Run the gel at 150 V for 1 h . Pro-Tip If the samples are running unevenly and...Add 20 mL of SimplyBlue SafeStain and incubate for 1 h with gentle agitation on a rocking platform. Pour...sink. Add 100 mL of deionized water and incubate for 1 h with gentle agitation on a rocking platform. Pour...

  6. Immunocytochemistry

    Type
    Protocol
    ...Last Update: January 20, 2022 Workflow Timeline Day 1: Seed cells Day 3-4: Fix and label cells Equipment...into 5 mL PBS. Protect from light. Procedure Section 1: Seeding cells Place a sterile poly-D-lysine coated...concentration will vary but generally ranges from 1-10 µg/mL. Add 500 µL of the diluted antibody to the...concentration will vary but generally ranges from 1-10 µg/mL. Add 500 µL fluorescently-labeled secondary...with a laboratory wipe to remove excess liquid. Add 1 drop of anti-fade mounting medium to the microscope...This protocol describes the basic steps for fixing and staining cells in culture with a primary antibody...antibodies to detect antigens in cells. Here we describe the basic steps for fixing and labeling cells ...

  7. Protocol - Over-Agar Antibiotic Plating

    Type
    Protocol
    ... resistance) Procedure Day 1 Prepare carbenicillin to a concentration of 1 mg/mL – 4 mg/mL in LB medium...lawn of E. coli and no apparent selection. 150 µL of 1 mg/mL Carbenicillin plated over-agar Plate shows several...over-agar Plate shows less individual colonies than the 1 mg/mL plate and effective selection. 150 µL of 4 mg...several individual colonies with smaller size than the 1 mg/mL and 2 mg/mL plates and effective selection. ... Bacterial Culture Introduction This protocol describes methodology for plating antibiotic over-agar for...

  8. Protocol - Bacterial Transformation

    Type
    Protocol
    ...get more colonies if you use 1 μl of a 1:5 or 1:10 dilution rather than 1 μl directly....and then (optional) incubate in 37°C incubator. Mix 1 - 5 μl of DNA (usually 10 pg - 100 ng) into 20-50 ...shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30...Because of this, nearly all plasmids (even those designed for mammalian cell expression) carry both a bacterial...

  9. Protocol - How to Ligate Plasmid DNA

    Type
    Protocol
    ...situations where the 3:1 ratio is not working or when doing more complicated cloning. While 3:1 will get you in...the insert is smaller than the vector) a 3 insert : 1 vector molar ratio will work just fine. We recommend...reagents Optimizing the Vector:Insert Ratio Although a 3:1 insert to vector ratio is usually sufficient, you ...phosphodiester linkages, which permanently join the nucleotides together. After ligation, the insert DNA is physically... likely to ligate to itself rather than to the desired insert. If you are in this situation, it is important...reactions. Because ligase buffer contains ATP, which degrades upon freeze/thaw cycles, it is a good idea to ...

  10. Protocol - How to Run an Agarose Gel

    Type
    Protocol
    ...Biolabs B7022S Procedure Pouring a Standard 1% Agarose Gel: Measure 1 g of agarose. Pro-Tip Agarose gels are.... Note: Loading buffer serves two purposes: 1) it provides a visible dye that helps with gel loading and...different buffers and do not use water). Microwave for 1-3 min until the agarose is completely dissolved (but...the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration and ...be pushed away from the well comb or towards the sides/edges of the gel with a pipette tip. Place newly...Always Run to Red. Turn OFF power, disconnect the electrodes from the power source, and then carefully remove...-30 mins, replace EtBr solution with water and destain for 5 mins. Using any device that has UV light,...
Showing: 21 - 30 of 40 results