HC 70 AL Lab Handout Polymerase Chain Reaction PCR

1
HC 70 ALLab HandoutPolymerase Chain
Reaction(PCR)

• Week 1
• Thursday
• April 2, 2009
• Daisy Robinton

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

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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?
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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?
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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?
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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?
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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!)