PCR Flashcards
enumerate the steps of PCR
- denaturation
- annealing
- extension
this step involves heating the reaction mixture to — the DNA, separating the double-stranded into single strand. what is the temp for this step?
DENATURATION
94-98C
the temperature is lowered around — to allow primers to anneal (bind) to their complementary sequences on the single stranded DNA template.
50 - 65 C
the temperature is raised to —, and the DNA polymerase extends the primers by adding nucleotides, thus — new DNA strands
72-75 C
synthesizing new DNA strands
the steps of PCR are repeated for — to achieve a significant amplification of the target DNA
20-40 cycles
maxter mix iincludes all the reagents required for PCR such as — enumerate!
- DNA template
- primers
- DNA polymerase
- nucleotides
- buffer
enumerate the key condiserations in preparation of PCR master mix
- reagent concentrations
- total reaction volume
- mix preparation
total volume of PCR is typically —
20-50 uL
enumerate the principles in primer design (6)
- primer length
- GC content
- melting temp
- avoiding self complementarity
- specificity
- GC clamp
primer length
18-24 nucleotides
GC content
40-60%
melting temperature
2C each other
adding a GC-rich region at the 3’ end can enhance?
primer stability
factors needed to be considered in PCR reaction (6)
- template DNA
- cycling conditions
- positive and negative controls
- reaction volume
- thermal cyclers
- data analysis
preferred template DNA
purified DNA with minimal contaminants
