C3: Biology Lab Techniques

Question 1

what is an experimental control?

Answer 1

when you do the same experiment but change one thing

Question 2

what is a positive control?
- negative control?

Answer 2

• a sample/test that should work and give a positive result (it will work)
• a sample/test that wont work and should give a negative result (it will fail)

Question 3

• what is a false positive?
• example?
• what is a false negative?
• example?

Answer 3

• when a sample gives a positive result when it shouldnt have, it was actually negative
• ex: a positive pregnancy test when a woman is not pregnant
• when a sample gives a negative result when it shouldnt have, it was actually positive
• ex: a person tests negative for HIV when they actually do have HIV

Question 4

what did the chargaff experiment say?

Answer 4

DNA from any cell has a 1:1 ratio of pyrimidines to purines

Question 5

what did the wilkins and franklin discover?

Answer 5

produced an xray image of DNA showing sugar phosphate backbones and nitrogenous bases

Question 6

what did watson and crick discover?

Answer 6

DNA is double helix, sugar phosphates are antiparallel

Question 7

what did griffiths do?

Answer 7

worked with 2 strains of bacteria streptococcus pneumonia where one was harmful and the other was not. learned that cell extracts from one bacteria can transform another bacteria, allows non-virulent bacteria to become virulent

Question 8

what did avery, mccarty, and macleod discover?

Answer 8

continued the work of griffith, discovered destroying DNA prevented virulence and is therefore the active agent in bacterial transformation

Question 9

what did hershey and chase discover?

Answer 9

that DNA was the active chemical in e. coli, radioactively tagged proteins in it and realized virus DNA enters the bacteria host cell while protein does not so therefore; DNA must be the molecule carrying genetic info

Question 10

what are the 2 forms in which cell lysis can happen experimentally?

Answer 10

1. solution/reagent based: cell of interest is isolated, detergent is added, cell lysis occurs, uninterested stuff is removed, molecule of interest (lysate) is isolated
2. physical disruption: cell tissue cells are disrupted by physical interruption (like shaking test tube), molecule of interest is exposed (blended)

Question 11

why is cDNA easier to work with over genomic DNA?

Answer 11

cDNA is shorter than mRNA and does not contain regulatory segments or introns

Question 12

what are restriction enzymes?
- what is their role in bacteria?
- what are they used to make?

Answer 12

bacterial enzymes that cut double stranded DNA at specific sites called recognition sites
- to destroy viral DNA which gets injected into the cell, restrict the reproduction of hostile viruses
- used to make plasmids and other types of recombinant DNA

Question 13

• what is a nuclease?
• endonuclease?
• exonuclease?

Answer 13

• an enzyme which cuts nucleic acids
• cut in the middle of the DNA chain
• nibble nucleotides from the ends of DNA

Question 14

• what are plasmids?
• what type of organisms are they found in?
• what is an important gene they contain?

Answer 14

• used to transfer and replicate DNA
• bacteria
• an antibiotic resistance gene like Amp which allows bacteria within the plasmid to survive when a selection agent like ampicillin is added to the growth culture

Question 15

• what are common selection agents used in plasmids for prokaryotes?
• eukaryotes?

Answer 15

• ampicillin, penicillin, streptomycin, tetracycline
• neomycin, puromycin

Question 16

• what is a polymerase chain rxn?
• what are the 3 stages of PCR?

Answer 16

• when a small amount of DNA can be amplified to millions of copies to study it
• 1. denaturation: 2 DNA strands are separated, H bonds separate
     
     2. annealing: primer binds to strand
     3. elongation: taq pol is heat resistant so it elongates the strands

Question 17

what is gel electrophoresis?
- DNA and RNA are ____ (negative/positive) because of _____ (deprotonated/ protonated) phosphates in the backbone
- ____ are denatured and reduced before running them on a gel, they’re ____ (negative/positive)

Answer 17

used to separate biological macromolecules based on size and charge. large molecules get stuck in the gel and move slowly (stay near the top), small molecules move quickly through the gel (found near the bottom)
- negative; deprotonated
- proteins; negative

Question 18

what is molecular cloning?

Answer 18

generating recombinant DNA and replicating it in a host organism

Question 19

what is transformation?

Answer 19

getting a piece of DNA into a prokaryotic cell

Question 20

what is transfection?
- is this easier/harder than transformation?

Answer 20

getting a piece of DNA into a eukaryotic cell
- easier since eukaryotes dont have cell walls (some)

Question 21

describe the process of screening and selection of bacteria once you get a plasmid into the cell

Answer 21

you first select the bacteria for the plasmid you want by using a plate with ampicillin to ensure only the bacteria you want will survive.
then, you conduct additional screening to make sure the bacteria has the correct plasmid

Question 22

describe western blotting

Answer 22

first cell lysis and protein denaturation occur, then the sample is prepared. each well contains all the proteins that were in the cell and then the gel is transferred to a membrane to ensure it does not degrade. then, blocking occurs to block the stickiness from the gel so that only the protein of interest is transferred. then, a series of enzymes (antibodies) detect this protein and analyze it

Question 23

what is ELISA?

Answer 23

enzyme linked immuno-sorbent assay, a technique that uses antigen antibody interactions to determine the presence of something in serum

Question 24

what is immunohistochemistry?
- describe the process

Answer 24

the study of expression of tissue
- 1. tissues are harvested in wax
2. thin slices of tissue are cut off and laid on a microscope slide
3. wax is melted off
4. antibodies specific for the protein of interest are added
5. samples are washed between steps
6. signal is detected via color change

Question 25

what is immunoprecipitation?

Answer 25

a way to isolate a protein of interest from a lysate sample

Question 26

what is restriction fragment length polymorphism (RFLP)?

Answer 26

a type of DNA profiling or fingerprinting, relies on the fact each individual has a small set of specific variations in their DNA

Question 27

what is transcriptional profiling via microarray?

Answer 27

used to compare gene expression between different samples

Question 28

what is comparative genomic hybridization?

Answer 28

gives information on DNA dosage (gene amplification or loss)

Question 29

what kind of images do scanning electron microscopes produce?
- transmission electron microscopes?

Answer 29

• 3D
• 2D with high magnification

Question 30

what is centrifugation?

Answer 30

separates cell components using centrifugal force, based on size and density.

Question 31

what is cell fractionation?
- what is it AKA?

Answer 31

repeated centrifugation at progressively higher speeds, fractionates cell homogenates into separate samples
- differential centrifugation

Question 32

what is flow cytometry?

Answer 32

used to count the # of cells expressing the biomarkers you stained for

Question 33

what is gene targeting?

Answer 33

used to generate very specific model organisms