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    PCR Technique

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    muthuraja6728
    This document describes the PCR technique and gel electrophoresis process. The aim is to amplify a segment of DNA using PCR and observe the results through gel electrophoresis. PCR uses thermal cycling to replicate the target DNA segment millions of times. The amplified DNA is then separated using gel electrophoresis, where it is run on an agarose gel and observed using UV light to verify amplification. Key steps include setting up the PCR reaction, preparing the agarose gel, loading samples into the gel, running electrophoresis, and visualizing the DNA bands.

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    PCR Technique

    Uploaded by

    muthuraja6728
    127 views5 pages
    This document describes the PCR technique and gel electrophoresis process. The aim is to amplify a segment of DNA using PCR and observe the results through gel electrophoresis. PCR uses thermal cycling to replicate the target DNA segment millions of times. The amplified DNA is then separated using gel electrophoresis, where it is run on an agarose gel and observed using UV light to verify amplification. Key steps include setting up the PCR reaction, preparing the agarose gel, loading samples into the gel, running electrophoresis, and visualizing the DNA bands.

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    Pcr Technique

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    This document describes the PCR technique and gel electrophoresis process. The aim is to amplify a segment of DNA using PCR and observe the results through gel electrophoresis. PCR uses thermal cycling to replicate the target DNA segment millions of times. The amplified DNA is then separated using gel electrophoresis, where it is run on an agarose gel and observed using UV light to verify amplification. Key steps include setting up the PCR reaction, preparing the agarose gel, loading samples into the gel, running electrophoresis, and visualizing the DNA bands.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    127 views5 pages

    PCR Technique

    Uploaded by

    muthuraja6728
    This document describes the PCR technique and gel electrophoresis process. The aim is to amplify a segment of DNA using PCR and observe the results through gel electrophoresis. PCR uses thermal cycling to replicate the target DNA segment millions of times. The amplified DNA is then separated using gel electrophoresis, where it is run on an agarose gel and observed using UV light to verify amplification. Key steps include setting up the PCR reaction, preparing the agarose gel, loading samples into the gel, running electrophoresis, and visualizing the DNA bands.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    of 5

    PCR Technique

    AIM:
    To replicate DNA using a PCR thermal cycler and associated primers
    To learn how to load and run a gel electrophoresis
    To observe DNA banding produced through the process of
    electrophoresis and to
    compare the PCR sample to the gene ruler provided

    PRINCIPLE :
    The PCR techniques developed in the mid 1980's are used to amplify
    a segment of DNA from a few copies to thousands or millions of
    copies. To verify that the gene, or segment of DNA, has been
    amplified, it is separated into reaction products by agarose gel
    electrophoresis.In the DNA lab where the objective of the experiment
    is insertion of a resistant gene, the amplified DNA is ligated directly
    into a standard cloning vector and transformed into bacteria. Inside
    the bacteria, the recombinant plasmids are replicated to a high copy
    number. The transformed bacteria colonies are isolated and the
    presence of the insert DNA is verified by restriction mapping.
    Materials:
    PCR thermal cycler
    Gel Electrophoresis equipment
    UV transilluminator*
    Micropipettors and tips
    Reaction Mix
    PCR Template
    Gene Ruler
    Ethidium Bromide DNA gel stain
    Agarose
    TBE buffer

    PCR Technique

    Loading dye
    Tube racks
    10 ml and 100 ml graduated cylinders
    Ziploc bags (medium sized)
    Waste bags
    Tube racks
    10 ml and 100 ml graduated cylinders
    Ziploc bags (medium sized)
    Procedure:
    Day 1
    (allow 20 minutes to prepare samples for PCR machine if each group
    of students has their own supply of reaction mix and primer mix)
    Setting up the PCR reaction
    1. Label a 0.2 mL, thinwalled micro tube with the initials of your group
    and place in ice.
    2. Mix reagents gently by flicking with finger.
    3. Practice pipetting technique with water and paper towelling,
    measuring droplet sizes. (Be sure to depress the pipettor only to the
    first stop in order to withdraw the precise amount of
    mix.)
    4. Pipette into the tube:
    a. 7.5 ul of PCR reaction mix (includes buffer, dNTPs, enzyme)
    b. 7.5 ul of DNA template plus primer mix
    5. Keep tube on ice until ready to start the PCR program.
    6. Load tubes into PCR machine and start cycling program
    (cycling parameters have already
    been entered into the machine. Program can be left to run overnight.)
    7. Freeze the samples after the reaction is finished if samples are not
    to be used immediately.

    PCR Technique

    Day 2
    (allow 1.5 2 hours to prepare gel and observe results)
    II. Preparation of PCR Reaction for Gel Electrophoresis
    1. Pipette 10 ul of the PCR reaction and place in a separate tube
    2. Add 2.5 ul of loading dye (contains a dense chemical called
    Ficol) to the PCR reaction.
    3. (Low speed gel electrophoresis would provide more explicit
    results; however, the 30-40 minutes running time can be
    prohibitive.)

    III. High Speed Gel Preparation


    1. Mix 10 ml of 10 x TBE with 90 ml of tap water.
    2. Pour 80 ml of this solution into the supplied agarose.
    3. Swirl to mix.
    4. Microwave on high for approximately 30 seconds.
    5. Swirl again.
    6. Microwave again for another 30 seconds. Watch to ensure that
    mixture does not boil over!
    Solution should be clear and on the point of boiling when the agarose
    is completely dissolved. Wearing gloves continue with next step.
    7. Add 8 ul of Ethidium Bromide DNA gel stain (wear gloves as this is
    a bio hazard!) and allow gel to cool for a few minutes before pouring
    the gel in the gel box.
    IV. High Speed Gel Box Preparation / Analyzing the PCR Reaction by
    Gel Electrophoresis.
    1. Place metal electrodes in slots provided (Both electrode connectors
    should be at the top of
    the box. The migration will then be from left to right.)
    2. Check that the lid will fit later when it is
    time to run the gel.
    3. Place a comb in with the flat side of the comb facing the direction of
    migration (towards the

    PCR Technique

    positive red electrode). Ensure that the comb is close to, but not
    touching, the bottom of the gel box.
    4. Pour gel into the box after the solution has cooled for a few
    minutes.
    5. After gel has solidified, pour 40 ml of water over the gel and
    carefully remove the comb.
    6. Set the 2-20 ul pipette to 12 ul and pipette the reaction into a
    well on a 1% agarose gel.
    7. Load 6 ul of the Gene Ruler (DNA) Marker into a separate well.
    8. Run the gel at 250 V for 6 minutes (high speed system) or at 150
    V for 30-40 minutes (low speed system)*
    * Running a High Speed Gel
    1.Ensure connection between electrodes on lid of box
    and metal posts inside the box are correct. Be sure buffer or water is
    in the box. Plug in and turn on power pack. The switch is at the back.
    2. Press the On button on the front display (central button with
    horizontal line). Adjust voltage to 240 V and set time to 6 minutes.
    3. Press Run.
    9. Once gel has finished, remove it from the box and put it into a
    sealed Ziploc bag for viewing in the transilluminator.
    10. Visualize the Ethidium Bromide DNA gel stain (handle with gloves
    as this is a bio hazard), stained gel under UV light. N.B. Gels are best
    viewed on the same day they are run. The bands will diffuse if the gel
    is left overnight.

    PCR Technique

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