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We narrowed to 634 results for: des.2

Showing: 621 - 634 of 634 results

  1. Virus Protocol - Generating Stable Cell Lines

    Type
    Protocol
    ...Workflow Timeline Day 0: Seed and transduce Cells Day 2–3 (am): Remove media, replace with fresh media containing...Biological Safety Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container...cell death, the cell media should be changed every 2–3 days to maintain the dose of antibiotic, which may...confluent 10 cm dish can be expanded into two 75 cm 2 flasks, etc. Pro-Tip This selection method results...with Lentivirus You may also like... Viral Vector Guides Virus Blog Posts Mol Bio Protocols Viral Service...dilutions and pick the population that has the most desirable level of expression. Over time, transgene expression...

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

    Type
    Protocol
    ...cooling to room temperature (~45 minutes). Method #2 Place mixed oligos in a PCR tube. Place tube in a ...a thermocycler programmed to start at 95°C for 2 minutes. Then, gradually cool to 25°C over 45 minutes...vector with 0.75-6 ng of annealed oligos). Transform 2-3μL into your favorite competent bacteria and plate...oligo overlap cloning, you can design a set of oligos containing your desired restriction sites and add them...that will pay off for years to come. Design Briefly, we will design overlapping oligos that once annealed...procedure will simply differ in terms of primer design). Let's assume that your favorite vector has a ...digest of existing sites in the original vector. Designing oligos To add NdeI, PacI, AscI and MfeI sites ...

  3. CRISPR Library Amplification

    Type
    Protocol
    ... recover, set up overnight growth (Estimated time 2-3 hours) Transformation should be performed at the... day to ensure that growth times are limited. Day 2: Harvest cells and purify DNA (Estimated time 3-4 ... Tubes should contain a total of 5 mL (3 mL SOC + 2 mL transformed Endura from two separate transformations...has been absorbed by the agar. This usually takes 1-2 minutes. Critical Be careful not to rip or shred the...incubation if needed. Place 100 mL sterile LB at 4 ℃. Day 2 (morning) Before beginning, prechill at least four...pellet. The total weight of each pellet should be ~1-2 g. Pro-Tip Make sure to weigh the empty tube beforehand...library during amplification. This protocol is designed to be as general as possible but note that individual...

  4. Protocol - Bacterial Transformation

    Type
    Protocol
    ...each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30-60 ...cells you are using). Put the tubes back on ice for 2 min. Add 250-1,000 μl LB or SOC media (without antibiotic...instead of on ice Reduce step 4 from 20 - 30 mins to 2 mins on ice before heat-shock Shorten or skip the ...Because of this, nearly all plasmids (even those designed for mammalian cell expression) carry both a bacterial...

  5. Protocol - Over-Agar Antibiotic Plating

    Type
    Protocol
    ... plate. Incubate plates at 37 ℃ for 18 hours. Day 2 Observe plates for colony formation. Shown below are...individual colonies and effective selection. 150 µL of 2 mg/mL Carbenicillin plated over-agar Plate shows less...individual colonies with smaller size than the 1 mg/mL and 2 mg/mL plates and effective selection. Selection Curve... Bacterial Culture Introduction This protocol describes methodology for plating antibiotic over-agar for...

  6. Gamma-Retroviral Vector Guide

    Type
    Guide
    ...Retroviral Vector Design. In Retroviruses . Cold Spring Harbor Laboratory Press. NIH Bookshelf De Ravin, S. S...gamma-retroviral vectors, you need three plasmids (Figure 2): Transfer plasmid — contains transgene, sgRNA, or...for different cell types (known as tropism). Figure 2: Gamma-retroviral plasmids. Created with BioRender.com...PMID: 24501411 Gilroy, K. L., Terry, A., Naseer, A., De Ridder, J., Allahyar, A., Wang, W., Carpenter, E.... Science Guides Viral Vectors Gamma-Retroviral Vector Guide Gamma-Retroviral...been widely used in the research community for decades. Wildtype gamma-retroviruses have been engineered...with certain cloning methods (such as a Gateway destination vector), so be sure to confirm your chosen plasmid...

  7. Pouring LB Agar Plates

    Type
    Protocol
    ...chloride 12.0 g agar-agar 1 L Sterile H 2 O Sterile plates of your desired size - we usually use 60 mm x 15 ...indicated, the antibiotic powder can be dissolved in dH 2 O. *Carbenicillin can be used in place of ampicillin...low a concentration for selection. Negative Result 2: Neither Strains Grows If neither strain grows, it's...next to the flame and begin pouring. Measure your desired amount of agar with a pipete for the first plate...

  8. Molecular Biology Reference

    Type
    Guide
    ...table below. Figure 2: General structure of a plasmid. Plasmid Element Description Origin of Replication...unique restriction enzyme recognition site (Figure 2). These elements allow for the propagation of the ...pairs with G. The order of nucleotides makes up the genetic code and provides the instructions to make ...thymine in RNA molecules. Every 3 nucleotides (codons) in a DNA sequence encodes for an amino acid. The genetic...double-stranded helix. To create the double helix, the nucleotides on the opposing strands of DNA form hydrogen ...acids. Each amino acid is encoded for by three nucleotides, termed a codon. There are 64 codon combinations... for codon-amino acid pairs. Over the past two decades, researchers have also expanded the genetic code...

  9. Pipetting Protocol

    Type
    Protocol
    ...right. Pipette Dispense Volume P2 0.2 to 2 µL P10 1 to 10 µL P20 2 to 20 µL P100 10 to 100 µL P200 20 to ...maximum possible setting on the pipette, set the desired volume of the liquid on the pipette. Depending ...of the pipette. Select the pipette tip that is designed to fit your pipette. To load a tip onto a pipette...from the container making sure not to touch the sides of the container with the pipette. Discard the pipette...

  10. Protocol - How to Create a Bacterial Glycerol Stock

    Type
    Protocol
    ... overnight culture to 500 μL of 50% glycerol in a 2 mL screw top tube or cryovial and gently mix. Notes...plasmid DNA, the plasmid will already be in your desired bacterial strain and you will not need to obtain...

  11. Molecular Cloning Techniques

    Type
    Guide
    ...Read more in our Gateway Cloning blog post . Figure 2: Summary of Gateway cloning. Created with BioRender.com...for it to then be digested and ligated into your desired plasmid. It is important to note that restriction...enzymes that cut your insert or backbone at only the desired location for your cloning project. Restriction ...rapidly shuttled into any compatible Gateway destination vector, which contains attR sites, via LR clonase...bacterial recombination to easily move it into any destination plasmid that fits your experimental goal. Although... Gateway donor , Gateway entry , and Gateway destination vectors with different promoters, tags, selection...Any dsDNA fragments can be used, so if properly designed, any insert fragment (PCR product or synthesized...

  12. Protocol - How to Ligate Plasmid DNA

    Type
    Protocol
    ...10μL reaction for 5X buffer) 0.5-1μL T4 DNA Ligase H 2 O to a total of 10μL Notes: If the DNA concentrations...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 ...

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

    Type
    Protocol
    ...DNA concentration alone. One method is to conduct 2 ligations for each plasmid you are trying to create...restriction enzyme digest (subcloning), including design and experimental procedures. Protocols...easily move YGOI into a mammalian expression vector. Design (Choosing enzymes) Many DNA analysis tools, including..., but do not cut within your insert Are in the desired location in your recipient plasmid (usually in ...your recipient plasmid as well as a specifically designed test digest later to verify that the insert was... fear. You have other options, such as: Adding desired restriction sites to flank your insert : You can...restriction enzymes that cut within your insert. Adding desired restriction sites to your recipient plasmid : You...

  14. Plasmid Cloning by PCR (with Protocols)

    Type
    Protocol
    ...DNA concentration alone. One method is to conduct 2 ligations for each plasmid you are trying to create...plasmid of interest. For this example, we will describe how to copy a cDNA from one vector into a new ...(YGOI) for ligation into a recipient plasmid. Designing Primers for PCR Based Cloning The basic PCR primers...that: Do not cut within your insert. Are in the desired location in your recipient plasmid (usually in ...examine the DNA sequence that we want to amplify and design primers that will bind to and replicate it. The...ends of the ORF and how these are used for primer design: Because we are cloning an ORF, we want to clone...TAAGCAGAATTCATGTGGCATATCTCGAAGTAC-3'. For the Reverse Primer, the design is similar, but we need to use the reverse complement...
Showing: 621 - 634 of 634 results