Jacobs:Protocol Polymerase Chain Reaction (PCR) Set-up and DNA Agarose Gel

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Contents

Overview

PCR is used to amplify specific regions of DNA. It can amplify a single gene, parts of a gene or non-coding regions. We will use PCR to amplify the FAK and Cre sequences in mice.

Materials

  • DEPC-treated H2O
  • PCR 2X Master Mix(PCR 2X Master Mix contains DNA polymerase, deoxynucleotide triphosphates (dNTPs),divalent cations (magnesium) and buffer solution.
  • Oligos (primers)
  • DNA template
  • DNA ladder
  • Ethidium bromide (EtBr)
  • 0.5 ml PCR thin-walled tube
  • Pipetman
  • Filtered pipette tips
  • 1.5ml microcentrifuge tubes
  • 1% agarose/Tris-Borate EDTA (TBE)/EtBr gel
  • 1X TBE buffer
  • UV transilluminator
  • Ethidium bromide extractor

Procedure

  1. One person will prepare a master mix for the entire class:
    1. Label two microcentrifuge tubes: “FAK” and “Cre”
    2. In each labeled microcentrifuge tube, mix the following for each master mix :
  2. Each person will prepare four PCR reactions using DNA from two mice
    1. Label four 0.5 ml PCR thin-walled tubes: “648FAK”, “648Cre”, “54FAK”, “54Cre” along with your initials on each tube
    2. Add 24 μl of each master mix to the appropriate tubes: FAK or Cre
    3. Add 1 μl of appropriate DNA template from mouse #648 and #54 into each tube
    4. Vortex and centrifuge for a few seconds
    5. Load into thermocycler and run the “FAK” thermocycling program overnight
  3. PCR products will be stored at 4C

Run DNA agarose gel on Thursday:

Procedure

  1. A 1% agarose/TBE gel will be prepared ahead of time
  2. Place the agarose gel in gel box with wells on the right hand side
  3. Add enough TBE buffer to just cover the gel
  4. Load 7 μL DNA ladder into the first well
  5. Label two 1.5 ml microcentrifuge tubes: “648FAK+Cre” and “54FAK+Cre”
  6. Mix 10 μL of each of your PCR products (FAK and Cre) for one animal in corresponding appropriately labeled tube and add 3 ul 10X Blue Juice dye:
    1. Tube labeled “648FAK+Cre” will contain: 10 μL of 648FAK + 10 μl of 648Cre + 3 μl Blue Juice dye
    2. Tube labeled “54FAK+Cre” will contain: 10 ul of 54FAK + 10 μL of 54Cre + 3 μl Blue Juice dye
  7. Load 10 μl of each DNA mixture into separate wells (all groups will load their samples on the same gel)
  8. Keep track of which wells contain your samples
  9. Run gel at 100V for ~1 hr, be sure the blue gel front is moving toward the anode (+)
  10. Stop the gel before the blue gel front runs off of the gel
  11. Remove the gel from the gel box wearing gloves (leave the TBE buffer in the gel box in case we have to run the gel longer)
  12. Place gel on UV transilluminator for visualization of DNA bands and note size of bands
    1. FAK2/3 PCR Products:400 bp band = Floxed FAK allele,290 bp band = Wildtype FAK allele
    2. Cre PCR products: 800 bp band = alpha1(I)Col (2.3kb)-Cre
  13. Run excess TBE buffer through an ethidium bromide extractor before disposing of it down the sink
  14. Store used extractor wrapped in plastic and log the volume of flow through.

References

Contact

  • History: CRJ-ABC, last updated 8/15/07


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