Team:Mississippi State/Doing PCR

From 2008.igem.org

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.
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