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

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:
 * 2) Label two microcentrifuge tubes: “FAK” and “Cre”
 * 3) In each labeled microcentrifuge tube, mix the following for each master mix :
 * 4) Each person will prepare four PCR reactions using DNA from two mice
 * 5) Label four 0.5 ml PCR thin-walled tubes: “648FAK”, “648Cre”, “54FAK”, “54Cre” along with your initials on each tube
 * 6) Add 24 μl of each master mix to the appropriate tubes: FAK or Cre
 * 7) Add 1 μl of appropriate DNA template from mouse #648 and #54 into each tube
 * 8) Vortex and centrifuge for a few seconds
 * 9) Load into thermocycler and run the “FAK” thermocycling program overnight
 * 10) 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:
 * 7) Tube labeled “648FAK+Cre” will contain: 10 μL of 648FAK + 10 μl of 648Cre + 3 μl Blue Juice dye
 * 8) Tube labeled “54FAK+Cre” will contain: 10 ul of 54FAK + 10 μL of 54Cre + 3 μl Blue Juice dye
 * 9) Load 10 μl of each DNA mixture into separate wells (all groups will load their samples on the same gel)
 * 10) Keep track of which wells contain your samples
 * 11) Run gel at 100V for ~1 hr, be sure the blue gel front is moving toward the anode (+)
 * 12) Stop the gel before the blue gel front runs off of the gel
 * 13) 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)
 * 14) Place gel on UV transilluminator for visualization of DNA bands and note size of bands
 * 15) FAK2/3 PCR Products:400 bp band = Floxed FAK allele,290 bp band = Wildtype FAK allele
 * 16) Cre PCR products: 800 bp band = alpha1(I)Col (2.3kb)-Cre
 * 17) Run excess TBE buffer through an ethidium bromide extractor before disposing of it down the sink
 * 18) Store used extractor wrapped in plastic and log the volume of flow through.

Contact

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

or instead, discuss this protocol.

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