T5 DNA Amplification (PCR) Flashcards

Flashcards about DNA Amplification (PCR)

What parts of the cell must be broken down to extract DNA?

To extract DNA, both the cell membrane and nuclear membrane must be broken down. This is done using a lysis buffer.

Where is the DNA after centrifuging the neutralized fish tissue lysate?

Supernatant

Where is the DNA after centrifuging the supernatant through the column?

Bound to column

Where is the DNA after centrifuging the wash solution through the column?

Still on column

Where is the DNA after centrifuging the elution solution through the column?

In the flowthrough (collected DNA)

How do researchers target the portion of DNA to be amplified during PCR?

They use primers, which are short DNA sequences that bind to the beginning and end of the specific DNA region to be copied.

Do you expect the pCOI plasmid to generate a PCR product? What about the negative control? Why or why not?

The pCOI plasmid should produce a PCR product because it contains the target DNA. The negative control should not produce a product since there is no DNA to amplify.

What should the results look like for each sample in the gel?

A successful PCR sample will show a band at 650 base pairs (bp). There may also be a smaller band below 100 bp, which is caused by primer dimers.

Why is it necessary to have PCR products purified before sequencing?

Purification removes leftover primers, enzymes, and unwanted fragments that could interfere with sequencing.

What primers will be used to amplify your PCR product?

The primers used for amplification are included in the Bio-Rad Master Mix and are designed to amplify the barcode region in fish DNA.

What steps of the process may impact the quality of the sequence that will be generated?

1. DNA preparation – poor samples reduce quality. 2. Purification – leftover impurities can interfere. 3. Amplification (PCR) – errors can lead to wrong or weak products. 4. Sequencing – machine or input errors can affect accuracy.

Where are the sequencing primer sequences on your PCR product? How did they get there?

They are located at the 5′ and 3′ ends of the target DNA sequence. They got there when the amplification primers were added during PCR.

What two aspects of primer design were used to ensure successful amplification from many fish species?

1. Primers are long enough to allow for mismatches between species. 2. They target a conserved region of DNA that is similar across many fish species.