MOL BIO LAB L7 (Midterms- PCR) Flashcards
A fundamental technique in molecular biology and genetics.
PCR
A widely used molecular biology technique for amplifying DNA. The procedure involves a series of temperature cycles, typically consisting of denaturation, annealing, and extension steps.
PCR
The initial step of Denaturation involves heating the reaction mixture around _____ to denature the DNA
94- 98C
The temperature is lowered to around ______ to aIIow the primers to anneal (bind) to their complementary sequences on the single-stranded DNA template.
50- 65°C
These steps (denaturation, annealing, and extension) are typically repeated for ______ to achieve a significant amplification of the target DNA.
20-40 cycles
To setup a PCR reaction, a _______ is prepared.
PCR master mix
In extension, the temperature is raised to_______, and DNA polymerase extends the primers by adding nucleotides, thus synthesizing new DNA strands.
72-75°C
Enumerate the reagents included in the master mix
DNA template, primers, DNA polymerase, nucleotides, and buffer.
The precise composition of the master mix depends on the?
specific PCR protocol and reagents used.
These are needed to be calculated to optimize the PCR reaction
- Concentration of primers
- DNA template
- dNTPs (deoxynucleotide triphosphate)
- Buffer components
The total volume of the PCR reaction depends on the type of PCR machine used but is typically?
20-50 uL
The master mix is prepared in a _______ to avoid contamination. It’s crucial to keep reagents on _____ and to use dedicated pipettes and tips to prevent cross-contamination.
sterile environment; ice
Primers should not form _________ or ___________
secondary structures; self-complementarity
Primers are typically _______ nucleotides in length.
18-24
The GC content should be around _____ to optimize primer annealing.
40-60%
The melting temperature of primers should be similar,
typically within?
2°C of each other
Primers should be designed to specifically anneal to the _________, avoiding non-specific binding.
target DNA
Adding _______ region at the ________ can enhance primer stability
GC-rich region; 3’ end
During the PCR reaction, what factors are needed to be considered?
- Template DNA
- Cycling Conditions
- Positive and Negative Controls
- Reaction Volume
- Thermal Cyclers
- Data Analysis
TRUE OR FALSE: Use a reliable thermal cycler that can accurately control weather and time machine
FALSE. It should be can accurately control temperature and time cycles
After PCR, the amplified products can be analyzed by _______, real-time PCR, or ________to verify the presence of the target
DNA.
gel electrophoresis; sequencing
NOTE: Keep in mind that PCR protocols and reagents may evolve, so it’s important to consult the latest literature and manufacturer’s instructions for the most up-to-date information and best practices
This is the type of DNA preferred as the template DNA
Purified DNA with minimal contaminants
These characteristics of template DNA are crucial (2pts)
Quality and Quantity
Include _________and ________in your PCR to ensure the reaction is working and to check for contamination.
positive; negative controls
Ensure that the _______ is consistent and that you are using appropriate reaction tubes or plates.
reaction volume
Series of Temperature cycles (3 pts)
- Denaturation
- Annealing
- Extension
Considerations in creating an effective primer design
- Primer length
- GC content
- Tm (Melting temp)
- Avoiding Self-Complementarity
- Specificity
- GC clamp
