quiz 1 : nucl/clon

what is the purpose of aseptic technique?

avoid contamination while making sure the right specimen reaches the right location

How is media sterilized?

Autoclave

using heat, pressure, and water (steam)

How can you control whether the media is solid or liquid?

By including agar

What is the goal of the streak plate?

Obtaining isolated colonies

what are the pros/cons of liquid broth?

pro: a lot of bacteria like to grow in broth, grows quickly

con: no isolation, can be hard to spot contamination

what are some pros/cons of solid media?

Pro: see different colony morphologies, quick growth, can see contamination

cons: can dry out, can hold limited sample volume

Draw and label a bacterial growth curve

what phase of growth do we want bacterial cells to be in for DNA extraction? Why?

Late log or early stationary

This allows for a high number of cells while avoiding DNA degradation

best for quantity and quality

what phase of growth do we want bacterial cells to be in for RNA extraction? why?

In the middle of log/exponenetial

RNA is not happening constantly

How could you determine what phase of growth your cells are in?

spectrophotometer

How can you determine if your broth culture is pure?

By using a streak plate and seeing if cultures are present

What lysis methods did we use in lab?

mechanical disruption from glass beads

What separation methods did we use in lab?

bind DNA to column

What DNA recovery method did we use is lab?

adding H2O, rinsing off the column

What method did we use to determine whether or not our experiment was successful? (DNA extraction)

nanodrop program

What methods can you use to determine if your DNA extraction was successful?

nanodrop, spectrophotometer, visually checking to see if clear or cloudy (not very reliable)

What do the different wavelength measurements mean, and what are the recommended amounts they should be at?

230 and 260 are used to measure purity

260 is used to calculate the quantity of DNA

260/280 is used to measure protein contamination ( should be greater than 1.8)

260/230 is used to measure salt/chemical contaminants (the higher the better, but should be greater than 2)

Which bond is the hardest to pull apart in nucleic acid? why?

G to C bond because it has 3 bonds

what are some cell types missing from this image?

Archaea, fungi, protists

which of these have cell walls? what are the cell walls made of?

animal cell- no cell wall (phospholipid bilayer)

plant cell- has walls (cellulose, other polymers)

bacteria- has cells (peptidoglycan)

archaea- has cell walls (various surface polymers, proteins, polysaccharides)

fungi- has cell walls (chitin)

protists- some have cells walls, some do not

List the different properties of gram positive and gram negative

gram negative: plasma membrane, peptidoglycan, outer membrane

gram positive: plasma membrane, thicker layer of peptidoglycan, no outer membrane

What is the first general step in DNA extraction?

Lysis

what type of cell would be the easiest to lyse? why?

animal cells

no cell walls

List detergents that are used in lysis, what did we use?

SDS, guanidinium salts, lysozome, and protienase K (protease)

we used guandinium salts

List forms of mechanical disruption

glass beads/bead beater, mortar & pestle/ liquid nitrogen, homogenizers, sonication, small gauge needles

What is lysozome? where can it be found? what does it do?

antimicrobial enzyme

can be found in human tears, breast milk, and chicken egg whites

it cleaves bacterial wall polysaccharides

what is peptidoglycan made of? what does this have to do with lysozome?

made of nam-nag

lysozome cleaves nam-nag linkages

What comes after lysis?

Separate DNA from proteins/ other cellular material

What comes after separating the DNA?

Recover DNA

List DNA extraction in order

Lysis

Separate DNA

Recover DNA

What are the different ways you can recover DNA?

column elution or ethanol precipitation

what are the pros and cons of organic extraction?

pros: cheaper

cons: takes a lot longer, more likely to get contamination

what is a part of organic extraction?

Lysis using SDS and Proteinase K

Separation with phenol chloroform

ethanol precipitation

What are some different methods of seperation?

cesium chloride density gradients

anion exchange separation

magnetic particle separation

describe the process of ethanol precipitation

add pure ethanol and salt

spin tube

clear pellet left at bottom (remove liquid)

add in lower ethanol

mix and spin tube

left with clear/small pellet of DNA

add buffer to dissolve (small volume)