Bio 333 Exam 1

How do you generate a molecular standard (λ / HindIII) ?

A molecular standard for DNA fragment sizes can be generated by digesting a known quantity of plasmid DNA with the restriction enzyme HindIII, resulting in predictable fragment sizes which can be used to assess the sizes of unknown DNA samples.

why do you generate a molecular standard (λ / HindIII)

Generating a molecular standard helps in determining the sizes of unknown DNA fragments by comparing them to the predictable sizes of the fragments obtained from the digestion of plasmid DNA with HindIII.

Making an agarose gel: Electrophoresis

the movement of a charged particle through an electrical field

Making an agarose gel: How did we make a 1% TBE agarose gel for DNA electrophoresis—what components were needed?

To make a 1% TBE agarose gel for DNA electrophoresis, we needed agarose powder, TBE buffer, and distilled water. A 1% TBE agarose gel is made by dissolving 1 gram of agarose in 100 ml of 1X TBE buffer, heating until clear, pouring into a tray with a comb, and allowing it to solidify.

Making an agarose gel: Why can you visualize DNA using EtBr?

Making an agarose gel: Why do you add a buffer before running the gel?

Making an agarose gel: How does DNA migrate to in an agarose gel, why (Toward what terminal (negative or positive)?

Making an agarose gel: Describes factors that affect DNA mobility through an agarose gel.

Making an agarose gel: Why do you add loading dye to each sample? How much and why that amount?

Making an agarose gel: What conditions can you change in the gel to change how fast (or slow) the nucleic acid moves through the gel?

Making an agarose gel: How do you resolve (more clearer see) two bands that are close in size?

DNA Restriction Digests: Describe the mechanisms by which restriction enzymes work.

DNA Restriction Digests: What are different types of restriction enzymes (general)?

DNA Restriction Digests: Know how to set up a restriction digest

• 1x Buffer (this means that if you have a 20ul reaction you add 2ul of 10x buffer)

• 1/10 the volume of enzyme (this means that if you have a 20ul reaction you add 2 ul of enzyme. Remember that enzymes are stored in glycerol so they do not freeze, but too much glycerol can inhibit reactions)

• DNA: typically about 1 ug

• Water: add to 20ul

Example:

Buffer 1 (10x): 2 ul

EcoR1: 2ul

DNA (1ug/ul): 1ul

Water: 15ul

Total volume: 20ul

DNA Restriction Digests: Name reasons why a restriction digest might not work.

DNA Restriction Digests: Write a palindromic sequence in DNA that is 6 letters long

Plasmid mapping: Know how to predict fragment sizes from a plasmid map (linear or circular).

Gel Extraction: Know how to figure out what type of enzyme you would use to excise a gene fragment from a plasmid

Gel Extraction: What is gel extraction and how to you perform a gel extraction? (you do not need to memorize the protocol)

Alkaline lysis (plasmid isolation from bacteria cells): What is the end product of the alkaline lysis procedure (mini preps)?

Alkaline lysis (plasmid isolation from bacteria cells): Know the purpose of re-suspension buffer

Alkaline lysis (plasmid isolation from bacteria cells): Know the purpose of lysis buffer

Alkaline lysis (plasmid isolation from bacteria cells): Know the purpose of precipitation salts

Alkaline lysis (plasmid isolation from bacteria cells): Why do you not get chromosomal DNA from am alkaline lysis procedure?

Alkaline lysis (plasmid isolation from bacteria cells): Why do you not get proteins from an alkaline lysis procedure?

Alkaline lysis (plasmid isolation from bacteria cells): Why do you not get RNA from an alkaline lysis procedure?

Alkaline lysis (plasmid isolation from bacteria cells): Why do you not get membranes from an alkaline lysis procedure?

Ligation: What is DNA ligation?

Ligation: What does DNA ligase do?

Ligation: What would happen if you forgot to add DNA ligase to your ligation mixture and why?

Ligation: In this experiment we essentially cut a gene (Kan resistance) from a plasmid and inserted it into the pAMP plasmid. What was the importance of cutting both plasmids with HindIII and BamHI. Why does this produce the desired fragment?

Making chemical competent cells: What is the purpose of adding Magnesium and Calcium Chloride to the bacterial cells during this procedure?

Making chemical competent cells: What is transformation?

Making chemical competent cells: Why do we perform a heat shock. Why do we grow the bacteria for an hour after the heat shock?

Making chemical competent cells: What is the mechanism of action of ampicillin and kanamycin. What do the resistance genes do to block these antibiotics.

Making chemical competent cells: What are microsatellite colonies and why do they form?

Making chemical competent cells: How do you know if the bacteria took up the plasmid?

Blue – white screening: What is the purpose of X-gal?

Blue – white screening: What is amp^R

Blue – white screening: Cells that take up (transform) pBlu turn what color? Why?

Blue – white screening: Cells that do not transform with pBlu stay what color? Why?

Blue – white screening: What color of cells should only be present on pBlu amp⁺ plates? Why?