Cell Bio Lab #3 - DNA Isolation and Gel Electrophoresis - Review

It is a chelating material used to purify other compounds via ion exchange. It does not bind to DNA, but it binds to ions like Mg2+ which are necessary for the enzyme nuclease to function so that it can dissolve the contents of the nucleus and put them into solution. Chelex also protects DNA from the effects of heating which are used to release DNA from the cells. It binds to transition metal ions which can damage DNA at high temperatures, and the ions that Chelex binds to can inactivate nuclease.

What is the role of Chelex 100 in DNA extraction?

Immortalized cells

This term describes cells that do not die and never stop dividing. Cancer cells and stem cells are examples of these

Ethidium bromide

This is a colored dye used in DNA gel electrophoresis that stains DNA and forms visible bands in the gel

A260/A280 refers to the ratio of DNA and RNA to proteins for assessing purity. For a pure sample, the value should be between 1.8 and 2.0, but if the value falls below that, there are protein impurities present. If the ratio is above 2.0, there is extra DNA

What does the ratio A260/A280 mean for a DNA purity assessment?

A260/A230 refers to the ratio of DNA and RNA to other impurities such as salts in the sample. For a pure sample, values should be between 1.8 and 2.0, so if it is less than 1.8, there are numerous impurities (likely to be salts) present. If the ratio is above 2.0, there is extra DNA.

What does the ratio A260/A230 mean for a DNA purity assessment?

We worked with breast cancer cells, MCF-7, which are early stage tumorigenic cells

What cells did we analyze in lab?

DNA and RNA

A260 is used to measure the presence of what?

Proteins

A280 is used to measure the presence of what?

Impurities (salts)

A230 is used to measure the presence of what?

Absorbance (optical density), agarose gel electrophoresis, or fluorescent DNA-binding dyes

What are the methods for assessing DNA yields>

Cell signaling pathway, disease pathogenesis, and drug discovery and action in vitro

Immortalized cell cultures are common ways of studying what?

HeLa Cells

These cell lines from Henrietta Lacks' cervical cancer were the first immortalized cell cultures to be used in scientific research