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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 40 results for: des.1

Showing: 31 - 40 of 40 results

  1. 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...bottle for autoclaving. We make 400 mL of agar in 1 L bottles and 200 mL of agar in 500 mL bottles. The...measure out 100 mg of ampicillin powder, add it to 1 mL of water, dissolve by vortexing, and filter sterilize... You should not store your plates for longer than 1 month at any temperature and should always check for... for contamination prior to use. Negative Result 1: Both Strains Grow Assuming the appropriate strains...next to the flame and begin pouring. Measure your desired amount of agar with a pipete for the first plate...

  2. Plasmid Cloning by PCR (with Protocols)

    Type
    Protocol
    ...has error rates that range from roughly 1 per 500bp to roughly 1 per 10 million bp depending on the polymerase...recipient plasmid to insert ratio of approximately 1:3. Since the number of base pairs for each varies,...cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent cells...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...

  3. Pipetting Protocol

    Type
    Protocol
    ...dispense small amounts of liquid (think: 0.1 µL to 1 mL). When working in a laboratory, properly dispensing...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 ...and the third (red) digit is hundredths. Therefore, 1 µL would read as 100 (as shown in the picture above...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...

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

    Type
    Protocol
    ...annealing can be achieved by one of two methods: Method #1 Place the mixed oligos in a 1.5mL microfuge tube. ...vector in molar ratios (vector:insert) between 4:3 and 1:6 in a standard ligation reaction (ex. to ligate an...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 ...between the EcoRI and SalI sites of the vector, we design a top oligo with each of the additional sites in...

  5. Using a Light Microscope Protocol

    Type
    Protocol
    ...Figure 1 depicts an image of a compound light microscope with the main components labeled: Figure 1: Diagram...magnifies it 10 times, and so on. The ocular lens also provides magnification and the power should be provided...provided on the microscope; often this lens provides 10x magnification. To determine the final magnification...that object! Equipment Light Microscope Prepared Slides (or other sample that can fit on a microscope stage...eyepiece so that you can see your image through both sides. Once you are happy with the lighting, use the coarse...

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

    Type
    Protocol
    ...recipient plasmid to insert ratio of approximately 1:3. Since the number of base pairs for each varies,...cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent cells...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...

  7. DNA Quantification

    Type
    Protocol
    ... measure your samples, place 1-2µL of mini-prepped DNA onto the pedestal. Close the lid and click measure...Repeat for each sample. Notes: Keep in mind that despite the accuracy of the NanoDrop, if two consecutive...

  8. Water Bath Protocol

    Type
    Protocol
    ...quicker. Pro-Tip Set up the water bath 30 minutes to 1 hour before you need to use it to allow the temperature... of bacteria or fungi. There are disinfectants designed to be used in water baths with instructions on...bath on and set the appropriate temperature as described in your protocol. How to set the temperature will...Set up your water bath so that it will reach the desired temperature by the time you need it for your experiment...water bath to prevent evaporation and maintain the desired temperature. This also helps the water bath heat...water bath even if you are using disinfectants as described above in step 3. You will also need to maintain...

  9. Gibson Assembly Protocol

    Type
    Protocol
    ...ssDNA. (Rabe & Cepko, 2020). Incubate the mix for 1 hour at 50 °C or follow manufacturer's instructions... Procedure Design your plasmid and order primers (see figure to the right). When designing your plasmid...and enrich for correctly assembled plasmids by designing primers to split an antibiotic resistance gene...and yield. If there are significant amounts of undesired product, gel-purify DNA segments. Otherwise, PCR... High-Fidelity DNA Polymerase - incorporates nucleotides to “fill in” the gaps in the annealed DNA fragments... (<150 bp). Pro-Tip Please note that the way to design the “stitching” primers and the amounts of primers...window) . NEB has other resources, such as a primer design tool (Link opens in a new window) . Gibson Assembly...

  10. Handling Plasmids from Addgene - Purifying Plasmid DNA

    Type
    Protocol
    ...solution, add 2-2.5 volumes 95% or 100% ethanol and 1/10 volume of 3 M Na-acetate (pH 4.8). Invert the microfuge...Last Update: Feb. 8, 2018 Equipment Desktop microcentrifuge Desktop vortexer Vacuum (optional) Reagents... several μg/μL. The protocol below is meant to describe the general procedure for purifying plasmid DNA...
Showing: 31 - 40 of 40 results