Lab Quiz 4 Flashcards
What does PCR stand for?
Polymerase Chain Reaction
What is the objective of PCR?
We want to amplify a gene in vitro, and implant it into bacterial vector, and hopefully get it back into the bacterial cell - transformation.
Why is it not advantageous to use normal DNA Polymerase?
Because it would make things very tedious - the DNA Poly would have to be added after the denaturation of the strand.
Instead, which type of DNA polymerase is used?
Taq Polymerase
What temperature does Taq Polymerase operate at?
72 Celsius
What is the first step of PCR?
Denaturation, usually around 95 degrees celsius
What is the second step of PCR?
The second step is annealing.
This refers to the primers, which are chosen specifically for the gene that you want to amplify.
Explain the primers and what they are used for in the last step of the cycle.
The primers include the forward and the reverse primer.
In the first cycle of the reaction,
the forward primer will bind to one of the template strands, in front of the gene we want.
the reverse primer will bind to the other template strand at the end of the gene we want.
What is the third step in PCR?
Extension via Taq Polymerase
How do we get the specific sequence we want amplified?
The first replication does not give us the specific sequence we want - the strands contain the sequence we want but they also have other stuff we don’t want.
The second replication does much of the same, but since we now have four copies, two of them will be almost perfect, but too long on on strand.
The third replication is when we really start to see the sequence we want.
After many, many replications, there will be so much of the specific sequence, that we won’t even be able to find the longer stuff.
What temperature is the annealing step usually done at?
55 degrees Celsius
What is used to change the temps during the course of PCR?
Thermal Cycler
