Welcome to Benchling

Benchling is an intelligent research platform with tools for note-taking, molecular biology, and sample tracking.

Create an account

Email
Already have an account?
Benchling is actively tested against the latest versions of Chrome, Firefox, Safari, and Edge. **It doesn't look like your current browser is supported** - for more information, [click here](/browsers).
Monospace normal
Monospace bold
0 of 0
Advanced Settings
    SOP: PCR amplification of  plasmid DNA
    • Notes
    • Metadata
    • Relevant Items
    Editing disabled on read-only entry.

     
    SOP: PCR amplification of  plasmid DNA

    Friday, 4/26/2019Introduction1.- Purpose2.- Scope3.- Principle4- Associated Documents5.- Important information to be documented throughout this procedureTable 16.- Safety Considerations7.- Important Notes7.1 Before starting your experiment:Table 27.2 During your experiment:7.3 After your experiment:8.- Equipment and Reagents9.- ProcedureTable 3: Reagents for a Taq PCR reactionTable 4: Amplification protocol10.- Storage11.- References12.-Document amendment historyDocument Amendment History
    Friday, 4/26/2019
    1.- Purpose
    This SOP describes the  method used for the amplification of specific DNA fragments by the Polymerase Chain Reaction, using plasmid DNA as a template. The amplified products may subsequently be analysed for presence or absence and size in agarose gels. Following purification, these can be used in downstream assays, including  sequencing, ligation and transformation and restriction digestion.
    2.- Scope
    This method is applicable to plasmid DNA. This method uses Taq polymerase as an example.
    3.- Principle
    Plasmid DNA is used as a template for amplification by DNA polymerase. The DNA is first denatured by high temperature upon the break up of the hydrogen bonds that link the double helix DNA strands (Denaturation). The temperature is lowered and the primers flanking the DNA fragment to be amplified are annealed with the DNA template at the complementary sequences (Annealing). The temperature is then increased slightly, the primers extended by DNA polymerase and the region between the primers is synthesized (Extension/synthesis). The cycle is repeated several times to exponentially create copies of the DNA sample template.
    4- Associated Documents
    Preparation of primer stock solutions
    PCR purification
    DNA quantification using the Nanodrop
    Gel electrophoresis
    5.- Important information to be documented throughout this procedure
    6.- Safety Considerations
    The operator performing this procedure is responsible for their own safety and that of others in the laboratory. Consult the lab manager for any safety issues before, during, and after the procedure if necessary.
    7.- Important Notes
    7.1 Before starting your experiment:
    7.1.1   Determine how many reactions you will include in your experiment, including positive and         negative controls.
    7.1.2   Make sure you know which reagents you will use (eg brand, type, etc), their location (eg fridge, freezer, bench) and verify they are available.
    7.1.3   Verify you if it is necessary to make work solutions of your forward and reverse primers and if so,  make them prior starting your experiment.
    7.1.4   Do the required calculations for your reactions and verify there is enough volume of reagents for your use, according to table 3  in section 9 (Procedure).
    7.1.5   Make sure you know what PCR amplification program you will use for your experiment.
    7.1.6   Make sure you understand how to operate the thermocycler. Contact the Lab Manager if you do not know.
    7.1.7   Make sure to clearly label your PCR reaction tubes. PCR tubes are very small and it is difficult to write long words on their lid. In order to identify them, it is preferable to write single digits or letters, such as 1,2,3, A, B, C, etc. Therefore it is advisable to codify them, BUT you need to record the corresponding codes in benchling, as in the example in Table 2  below:
    7.2 During your experiment:
    7.2.1   Wear gloves to avoid contaminating the reaction mixture or reagents.
    7.2.2   Follow good micropipetting techniques and practices throughout the procedure.
    7.2.3   Taq DNA polymerase: Polymerases are extremely temperature sensitive. Make sure it is kept cold throughout the procedure. Only take out of cold storage immediately before use, and place back into cold storage immediately after use. Also, spin the vial briefly in a microcentrifuge before use to ensure that no reagent droplets remain in the lid.
    7.2.4   Verify you have included all the reagents in your PCR reaction tubes and all the volume was dispensed from the tip into the reaction mix
    7.2.5   Verify the required amplification program has been selected properly in the thermocycler and it started properly
    7.3 After your experiment:
    7.3.1  Make sure you return all used reagents and DNA template to the appropriate storage location.
    8.- Equipment and Reagents
    8.1 Thin walled 200 µl PCR tubes
    8.2   Racks capable of holding PCR and microcentrifuge tubes
    8.3 Pipettes for pipetting volume ranges from 0.25 μL to 50 μL
    8.4 Sterile pipette tips
    8.5   Microcentrifuge capable of holding 1.5 mL tubes and producing at least 17,900 x g
    8.6   Thermocycler
    8.7    Permanent marker
    8.8    PCR reagents as in table in section 9
    9.- Procedure
    9.1   Start by filling in the table of reagents that will be added to the reaction mixture as below
    9.2  Arrange all reagents needed in a cold block (except Taq DNA polymerase) and let them thaw completely before setting up a reaction. Keep the reagents on ice throughout the experiment. Gently vortex and briefly centrifuge all solutions after thawing
    9.3 Label the required PCR  tubes with permanent marker and include sample details or code, as in the example of table 3 above.
    9.4 Pipet the reagents following the order in table 3: water, PCR buffer, dNTPs, MgCl2 (may be already  in the buffer, verify the required final concentration for your experiment), forward primer, reverse primer, template DNA and DNA polymerase. Be careful to avoid contamination of your reagents, and DNA sources. When working with DNA and enzymes, it is highly recommended to not reuse tips.
    9.5 In a separate PCR tube add all the reagents with the exception of template DNA for a negative control (increase the water to compensate for the missing volume)
    9.6 When needed in your experiment, prepare in the same manner, a positive control reaction with template of known size and appropriate primers
    9.7 Gently mix the solution by “finger vortexing” (flick with finger several times), to allow dispersal of Taq DNA polymerase in the reaction mix. Care should be taken to avoid the formation of bubbles.
    9.8 Briefly spin down the PCR tubes (2-3 s) using a tabletop microcentrifuge in order to ensure that all of the reagents are in the reaction mixture
    9.9 Place the PCR tubes  into the selected thermocycler.
    9.10 Once the lid to the thermocycler is firmly closed, start the required amplification program, as in table 4.
    10.- Storage
    If the amplified DNA is NOT to be immediately used, label the tubes with your initials and date and temporarily store at 4°C or freeze at -20 °C for long-term storage in assigned storage box. Properly record  the storage location in the corresponding section in benchling.
    11.- References
    K. (1998). At the bench: A laboratory navigator. New York: Cold Spring Harbor Laboratory Press.
    12.-Document amendment history

    Welcome to Benchling!

    Sign in to view data.

    Split Workspace