Team:Mississippi State/Doing PCR

=Doing Pfx50 PCR=


 * The purpose of PCR is to hopefully amplify a specific DNA sequence by heating and cooling a DNA/Primer mix. The procedure is as follows:


 * 1) Prepare 10x dilution of cDNA sample: 1ul cDNA + 9ul ddH2O
 * 2) Prepare PCR mix: 38.5ul ddH2O + 5ul Pfx50 Buffer + 1.5ul dNTP mix + 1.5ul LiPA1 + 1.5ul LiPA2 + 1ul cDNA + 1ul Pfx50 = 50ul.
 * 3) Label "LiPA"
 * 4) hit vortex
 * 5) short spin
 * 6) put in PCR
 * 7) Select the Pfx PCR program
 * 8) Prepare 1% Agarose Gel: 0.25g Agarose + 25ml 1xTAE Buffer --> in flask, weigh then microwave until boil a few times --> cool until warm --> weigh again, adding ddH2O until original weight --> store at 55C.
 * 9) Pour gel into gel tray with template in place.
 * 10) Allow get to set.
 * 11) Remove template, pour 1xTAE until it reaches top of tray.
 * 12) Add 1ul DNA Ladder to 1st well
 * 13) Put 1ul dye on parafilm
 * 14) remove PCR product, prepare 1.5ml microtube labeled: "Date, IGEM, LiPA PCR, Sample no."
 * 15) Get 2ul PCR product, mix with dye.
 * 16) Pipette mix into next well.
 * 17) Put remaining PCR product into 1.5ml microtube at -20C
 * 18) Hook up Electrophoresis (Red(+) on right, black(-) on left)
 * 19) Set voltage to 80V for 60min.
 * 20) When gel finishes, turn off power, remove gel, take UV picture, check for results.