Lab Quiz I

Question 1

Restriction Enzyme Role in Bacteria

Answer 1

restriction enzymes cut foreign DNA such as viral DNA fragments of different lengths; cut DNA recognize certain sequences (endonuclease)

Question 2

Bacteria DNA Protection Against Restriction Enzyme

Answer 2

bacteria protect their own DNA by modifying bases usually methylation at the recognition sites

Question 3

Gel Eletrophoresis

Answer 3

Gel electrophoresis uses an electric field to drive negatively charged molecules through a gel that acts as a molecular sieve. Shorter DNA molecules are less hindered by the agarose matrix and migrate faster than longer DNA molecules

Question 4

Cloning Vectors

Answer 4

I. an origin of DNA replication so they can be maintain in a cell
II. a gene such as an antibiotic resistance gene to select for cells that carry the vector
III. a unique restriction site or series of sites into which a foreign DNA molecule may be inserted

Question 5

Antibiotic Resistance Gene

Answer 5

Foreign DNA are inserted into one of the unique restriction sites in the gene and transformed into E. Coli cells. Transformed cells are plated on a medium containing the appropriate antibiotic to select for cells that carry the plasmid

Question 6

Restriction Enzyme Site 4bp vs. 6pb

Answer 6

4bp will be closer spaced than the 6bp recognition sites; 4bp occur approximately 4^4 = 256 bp where was the 6bp sequence will occur with an average frequency of 4^6 = 4096

Question 7

Agarose Gel

Answer 7

I. separates DNA fragments based on their relative sizes

II. DNA negatively charged sugar phosphate backbone so moves toward positive pole

III. smaller DNA less bulky and can travel through gel matrix quicker

Question 8

Agarose Gel (Loading)

Answer 8

I. anchor loading arm on the table and keep steady with other hand

II. move the pipette tip over the center of the well to be loaded without jamming it inside

III. slowly released sample and let it sink in to the well

IV. do not release the plunger until you have withdrawn the pipette

V. place cover over the running tank and connect black-black electrodes and red-red
turn on power supply

Question 9

Mini Prep Procedure

Answer 9

I. centrifuge E. Coli cells to obtain a bacterial cell pellet and remove liquid and then add cell resuspension solution

II. add lysis solution and alkaline solution → lyse the cells causing cells to release content and denature both nucleic acid and proteins

III. add neutralization solution → genomic DNA and cellular proteins to precipitate out of solution but plasmid and cellular RNA molecules remain in solution and renature

IV. centrifugation and keep leave behind the pellet (denatured DNA and protein) and keep the supernatant (plasmid DNA)

V. pipet the supernatant into promega spin column and centrifuge which causes the DNA to bind to the inside of the column while bugger flows through

VI. Wash solution

VII.

VIII. Nan-Drop instrument

Question 10

Digest Plasmid Purpose

Answer 10

to confirm the identity of the plasmid that we isolated from the E. Coli bacteria using StuI enzyme and BamHI

Question 11

Yeast Model Organism

Answer 11

simple and inexpensive to grow and maintain and mutants are easy to isolate and characterize and yeast genes have mammalian homologs therefore provide a simple eukaryotic model for cellular processes

Question 12

CEN

Answer 12

mediates attachment of the plasmid to the mitotic spindle ensuring segregation of the plasmid to both the mother and daughter cells; centromere-like region where microtubules can bind to and pull

Question 13

ADE2 (Selectable Marker); Plasmid Integration

Answer 13

ADE2 present in 402 412 and 422 plasmids
ade2 mutants cannot grow on media lacking adenine (red)
when ade2 mutant yeast cells are transformed with one of the plasmids containing wild type ADE2 gene they will be able to grow on media lacking adenine cream color

Question 14

Gel Electrophoresis

Answer 14

The molecules to be separated are pushed by an electrical field through a gel that contains small pores. The molecules travel through the pores in the gel at a speed that is inversely related to their lengths. This means that a small DNA molecule will travel a greater distance through the gel than will a larger DNA molecule.
Because DNA and RNA are negatively charged molecules, they will be pulled toward the positively charged end of the gel.

Question 15

ARS

Answer 15

PRS412: replicated once before dividing and origin of replication for yeast; was first identified on yeast linear chromosome

Question 16

PRS402

Answer 16

5528bp

Question 17

PRS412

Answer 17

6042bp and CEN / ARS

Question 18

PRS422

Answer 18

6868bp and 2micron

Question 19

ADE2

Answer 19

selectable marker; a gene that encodes one of the enzymes for biosynthesis of adenine

Question 20

2 micron

Answer 20

PRS422: origin of replication for yeast and can be replicated at any time / multiple times

Question 21

Poly-cloning Sites

Answer 21

restriction enzyme sites or endonuclease sites: cut DNA at specific sequences

Question 22

AMPr

Answer 22

ampicillin resistant; selectable marker can survive

Question 23

Mini Prep Purpose

Answer 23

Free plasmid from bacterial host using mini-prep kit by promega

Question 24

Transformation Efficiency

Answer 24

the plasmid gets in to the cell that it can be copied and passed on to the daughter cell

Question 25

Segregation Efficiency

Answer 25

where segregation means the transmission of the plasmid from mother to daughter cell via mitosis and cell division

Question 26

Yeast Transformation Procedure

Answer 26

incubate and mix and then plate on adenine plate

Question 27

Transformation Purpose

Answer 27

introduce a foreign plasmid into bacteria, the bacteria then amplifies the plasmid, making large quantities of it

Question 28

Transition

Answer 28

interchanges of two-ring purines (A G) or of one-ring pyrimidines (C T)

Question 29

Transversion

Answer 29

are interchanges of purine for pyrimidine bases