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HC 70 AL Lab Handout P olymerase C hain R eaction (PCR)

Explore the fundamentals of Polymerase Chain Reaction (PCR), from primer design to gel visualization. Understand the stages, requirements, and precautions to ensure successful PCR outcomes.

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HC 70 AL Lab Handout P olymerase C hain R eaction (PCR)

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  1. HC 70 ALLab HandoutPolymerase Chain Reaction(PCR) Week 1 Thursday April 2, 2009 Daisy Robinton

  2. What is the Polymerase Chain Reaction? What is the main purpose of PCR? How does it accomplish this?

  3. What are the requirements for PCR? • Knowledge of DNA sequence • Why is this information necessary? • DNA template containing sequence of interest • Specific Primers • What is a primer? • Where do these primers come from? • DNA polymerase • What kind of polymerase is used in PCR? Why? • Where did this polymerase come from? • dNTPs (deoxyribonucleotide triphosphates) - What is the purpose of these? • Buffer and Salts (KCl, MgCl2) - What do we need these for? • Thermocycler - How does this regulate PCR?

  4. Where do the primers come from? How many primers do we need? Where will these bind? What considerations need to be made in when designing primers?

  5. What are the three stages of PCR? What bonds are broken during DENATURATION? What will the orientation of the primers be relative to the template DNA? What occurs during EXTENSION?

  6. Time  How does a Thermocycler regulate these stages? What is the ideal temperature range for each stage of PCR? Why does each stage have its specific temperature range? What is the purpose of the high temperature for a long period at the beginning of PCR?

  7. How do you visualize PCR products? What would you expect to see after running a gel? WHY? What information does the gel photo give you? What if you see more than one band in a lane? What can the ladder tell you?

  8. What general considerations need to made when performing PCR? PCR is very sensitive to contamination Wear gloves. Use filter PCR tips. NEVER use the same filter tip in different solutions. Use NEW solutions if you suspect they are contaminated. Check off each solution as you pipette it into each tube. Stay focused (no chit-chat!)

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