Polymerase Chain Reaction Flashcards
the initial step involves heating the reaction mixture to around 94-98 degree celsius to denature the DNA, separating the double stranded template into single strands
denaturation
the temperature is lowered to around 50-65 to allow the primers to anneal (bind) to their complementary sequence on the single stranded DNA template
annealing
what temp should the reaction mixture be heated
94-98C
temp used for annealing (temp is lowered)
50-65C
The temperature is raised to 72-75 and DNA polymerase extends the primers by adding nucleotides thus synthesizing new DNA strands
extension
in extension, temp is raised to ?
72-75C
PCR procedure are typically repeated for how many cycles to achieve a significant amplification of the target?
20-40 cycles
use of PCR
widely used molecular biology technique for amplifying DNA
amplifying DNA PCR procedure involves a series of temp cycles which typically consist of?
denaturation, amplification, extension
materials used for PCR (10)
- DNA template (provided)
- Forward and reverse primers (provided)
- PCR reagents (DNA polymerase, dNTPs, buffer)
- Microcentrifuge tubes
- PCR thermal cycler
- Electrophoresis equipment
- Agarose gel and buffer
- DNA ladder marker
- Gel electrophoresis staining solution
Pipettes and tips - Safety equipment (lab coats, gloves, goggles)
to set up a PCR reaction, a ___ is prepared
PCR master mix
the master mix includes all the reagents required for PCR such as
DNA template, primers, DNA polymerase, nucleotides, and buffer
key considerations for prep and calculation of PCR master mix
- reagent conc
- total reaction volume
- mix preparation
The total volume of the PCR reaction depends on the type of PCR machine used but is typically
20-50 pL.
master mix should be prep in what environment to avoid contamination?
sterile environment
where should reagents be kept
on ice
components of primer design
- primer length
- GC content
- Tm (melting temp)
- avoiding self complementarity
- specificity
- gc clamp
various factors that need to be considered for PCR reaction
- template DNA
- cycling conditions
- positive and negative controls
- reaction volume
- thermal cyclers
- data analysis
primers should typically be ___ nucleotides in length
18-24
the GC content should be around ___ to optimize primer annealing
40-60%
the melting temp of primer should be similar, typically within __ of each other
2C
primers should not form ___ or ___
secondary structures or self complementarity
how to enhance primer stability
adding a GC-rich region at the 3’ end
