bioz476: dna purification and electrophoresis

Flashcards covering DNA purification protocol using QIAquick kits, reagent functions, safety information, and gel electrophoresis.

What is the primary purpose of purifying a PCR product before sequencing?

To remove PCR reagents that are no longer needed, such as PCR buffer, Taq polymerase, primers, extra DNA, and dNTPs.

Under what chemical conditions does DNA bind to the silica membrane in a spin column?

DNA adsorbs to the silica membrane in the presence of high salt concentrations.

What conditions are required to achieve DNA elution from the silica membrane?

Low salt concentrations and basic pH.

What is the function and chemical composition of Buffer PB in the QIAquick procedure?

Buffer PB has an acidic pH to help remove extra material; it contains guanidine hydrochloride and isopropanol.

What is the role of Buffer PE during the DNA purification process?

It acts as a wash buffer to wash away small DNA fragments and leftover dNTPs.

What is the composition and function of Buffer EB?

Buffer EB is composed of $10\,\text{mM Tris Cl, pH } 8.5$ (a basic buffer) and is used to elute the DNA fragment.

How much Buffer PB is added to the diluted PCR reaction in step 3 of the purification protocol?

$$250\,\mu l$$

What volume of Buffer PE is added to the spin column during the wash step?

$$750\,\mu l$$

Why is the empty spin column centrifuged at top speed for $1\,\text{min}$ in step 10?

To dry the filter by removing residual wash buffer.

How long should Buffer EB sit on the spin column membrane before centrifuging to elute DNA?

$$1\,\text{min}$$

At what speed and for how long should the spin column be centrifuged during the DNA elution step, and why?

At $1/2$ speed for $3\,\text{min}$, because spinning at higher speeds will cause the lid of the $1.5\,\text{ml}$ tube to fly off and damage the centrifuge.

What gel concentration and buffer are used for running the PCR products during gel electrophoresis?

A $2\%$ agarose gel in $1\times\text{TAE}$.

How are the DNA samples prepared with loading buffer and water before loading onto the agarose gel?

Each sample ($5\,\mu l$ unpurified PCR, $10\,\mu l$ purified PCR, or $10\,\mu l$ DNA extract) is mixed with $2\,\mu l$ of loading buffer and $3\,\mu l$ of $\text{dH}_2\text{O}$.

What voltage and run time are specified for running the agarose gel electrophoresis?

$100\,\text{V}$ for $20\text{--}25\,\text{min}$.

What active hazardous components are contained in Buffer PN?

Sodium perchlorate and isopropanol.

What hazardous compound is contained in Buffer QG?

Guanidine thiocyanate.

What plant marker gene, animal marker gene, and animal band size are identified on this gel electrophoresis result?

The plant gene marker is $rbcL$, the animal gene marker is $COI$, and the animal band size is $580\,\text{bp}$.

According to this DNA fragment binding-size range diagram, what size range of DNA fragments is recovered by the QIAquick PCR Purification Kit?

Fragments from $100\,\text{bp}$ to $10\,\text{kb}$ are recovered, while smaller fragments from $10\,\text{b}$ to $40\,\text{b}$ are removed.