Laboratory quiz

• a260/a230

  2.0
• technique that amplifies a stretch of dna

  pcr
• requirements for pcr

  dna template

  dntp's

  taq polymerase

  primer

  buffer
• three steps of pcr

  denaturation

  annealing

  elongation
• tube is heated at 95 degrees to separate the double stranded dna

  denaturation
• primers bind to their complementary regions on the single stranded dna template

  tube is cooled to 45 degrees depending on primer sequence

  annealing
• polymerase uses annealed primers as starting point for dna synthesis

  temperature raised to 70 degrees which is ideal for taq polymerase

  elongation
• factors in designing a primer


  length of primer

  annealing and melting temperature

  gc content

  secondary structure
• 18 to 24 base pairs

  length of primer
• non-specific dna amplification products

  short primer
• slow hybridization rate

  long primer
• allows primers to base pair with dna

  annealing temperature
• half of primers dissociate from dna

  melting temperature
• 5 degrees below the melting temperature so that most primers bind to the template (could result in non specific binding)


  annealing temperature
• 40%-60%


  gc content
• gc count and position

  2-3 g's and c's to the 3' end of primer
• 3 hydrogen bonds which promote stronger specific binding to dna template which ensure that reactions work properly


  gc base pairs
• avoid formation of hairpins or dimers

  secondary structure
• too many repeats could cause mispriming and the primer can anneal to unintended locations

  secondary structure
• a260/a280

  1.8