Clemson Advanced Micro Lab Quiz 1

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Autoclaving

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100 Terms

1

Autoclaving

Use of high-pressure steam to sterilize liquids and certain labware items.

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121 C, 15 psi, 15 min/liter

minimum conditions for autoclaving

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Dry Heat

Ovens set to a minimum of 160C for 2 hours

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4

Filtration

Physical process that literally removes microbes from suspension.

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0.45 um, 0.22 um

Common pore size for filtration

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Incineration

Flaming of loops and other materials to sterilize prior to use

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30-300

target range for plate counts

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8

10^-6

So, if you are handed a culture and were told that it had 100,000,000 cfu/ml, what would your final dilution need to be to hit the range?

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9

Filter Sterilization

used for small to moderate volumes with the small volume method, syringe filter.

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10

Heat labile

What materials would you use filtration for instead of autoclaving.

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11

Broth

A liquid used to grow microbes

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12

Solid

broth with a solidifying agent

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13

Complex Media

solutions of material of unknown composition

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Defined Media

solutions which the concentration of all materials within are known

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Enriched Media

media used to encourage the growth of organisms with specific capabilities

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BSL1

Good sanitation, proper PPE, proper disposal of biological wastes, reduction of aerosols and other means of transmission

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Biosafety Cabinets

aka laminar flow hoods, use HEPA-filtered air to provide a curtain of protection from stray contaminants, used for BSL2 or higher

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Visual Observations

Partial sunlight, some turbidity, decaying plant matter, healthy plant life

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Measured Observations

pH, temperature, salinity, turbidity, dissolved oxygen, nitrate, phosphate, total organic carbon, etc

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Fastidious

require extra nutritional support in the form of a growth factor

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Growth Factor

Salt, sugar, blood, chocolate (lysed RBC)

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Enrichment Parameters

pH, oxygen content, temperature, salt concentration, organic content

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No Universal Medium

You could probably think of all microbiological media as selective. Why?

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Increase cell abundance

What does starting with a liquid culture system do?

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plate, deep tube, broth, slant

Physical Growth Forms for media

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26

High Throughput culturing

Method to culture (partially) thousands of isolates

Make liquid extract from ecosystem Many small cultures (96 per plate) Automated

Allows for rapid assessment of biotech potential as well as the ability to acquire some physiological data

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Microcosms

Little Ecosystems, Highly varied

Design suited to system under study

Common materials used and equipment adapted as necessary

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r Strategists

grow faster

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k strategists

slower growth, grow to carrying capacity of land

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30

Spectroscopy

Use of light to measure chemical concentration and/or makeup

Based upon the interaction of specific wavelengths with materials

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Pathlength

Distance light travels through analyte

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Extinction Coefficient

The amount of a specific wavelength of light is absorbed by a standard concentration of a chemical. 1 M in 1 cm pathlength

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Beers Lambert Law

C = A/ε —> C is concentration, A is Absorbance (at X nm), and ε is the extinction coefficient at the defined wavelength.

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Uses of Spectroscopy

Counting cells, finding concentration via a standard curve, determining the chemical makeup

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Spectroscopy Strengths

not destructive, quick, easy to use, minimal data processing, inexpensive

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micro < preparative < ultra

Types of Centrifuge ranked smallest —> largest

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Proportional

Relationship between RPM & G-Force

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Precipitate (pellet)

at bottom, more dense

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Supernatant (supernate)

on top, less dense, liquid

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Preparative Centrifuge

Refrigeration is standard, allowing you to protect your samples from heat degradation.

The chamber is armored in case of rotor failure or operator error.

Excellent for separating modest amounts of cells from medi

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Fixed Angle Rotors

Holds tubes of 50 ml max volume.

The pellet will be at the bottom, towards the outside of the rotor.

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same orientation, balanced weights

How should you load a centrifuge rotor?

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Swinging Bucket Rotor

allows you to use gradients more easily to separate materials.

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Differential Centrifuge

To sort suspended particulates by size/density

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speed, temperature, time, buffer/gradient composition

How to report Centrifuge conditions

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xg, C

Units for speed, temperature of centrifugation

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47

polymerase chain reaction

what does PCR stand for?

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48

DNA primers, DNA polymerase, DNTPs, Buffer, DNA sample

Key components of PCR

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thermal cycler, PCR tubes

Equipment needed for PCR

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Denaturation, Annealing. Extension/Elongation, Final Elongation, Final hold

General steps of PCR

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Denaturation

DNA sample starts at room temp and heated

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Annealing

Primers bind to tell DNA pol where to start

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Extension/Elongation

DNA pol works - puts into place

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DNA purity, Phenolic Contamination

Two major issue with PCR success

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260/280 (DNA/Protein), 1-8-2.2 ratio

Goal for readings of DNA on spectrometer & ratio

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1 in 1 out, remove base, add x3

Three methods of site-directed mutagenesis

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manipulate DNA to add to it with recombinant protein to purify

Goal of protein tagging

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epitope tag

site for antibodies to bind

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protein will have affinity to stick to things

affinity tag

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sticks to specific parts of the chromatography column

chromatography tag

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qPCR

how much of X is in a DNA sample

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RT-qPCR

determining the amount of nucleic acid present in a sample uses fluorophores

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control DNA of the same size

requirement of qPCR

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Reverse Transcriptase PCR

technique used to monitor the presence of any RNA within the cell

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ssRNA —RT—> dsDNA —> PCR

Experimental design of Reverse Transcriptase PCR

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you need to know what you are looking for

Limit of Reverse Transcriptase PCR

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Methodology

different between RT-PCR and RT-qPCR

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live cultures, refrigeration, freezing, lyophilization

Methods of preservation

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70

Live cultures

not well preserved, requires periodic passage

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Refrigeration

live cultures, held at 4 C, periodically passaged

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Live cultures, Refrigeration, due to genetic change build up

Two least desirable methods of preservation and why

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Freezing

-80 to -120 C, addition of cryopreservative

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10-15% glycerol, prevent ice crystals

Suitable Cryopreservative & goal

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lysophilization

removal of water via sublimation

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solid —> gas

sublimation

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high vacuum, high SA to volume ratio , frozen material

requirement of lyophilization

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long process

limits of lyophilization

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gather/confirm quantitative data about a process & rates

why are assays necessary?

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80

Biological Assays

growth, no growth, follow cellular responses, high throughput analysis streams

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pH, temp, Ion concentration, reagent solubility/stability/aggregation, order of reagent addition, instrumentation

Things to consider in Biochemical assays

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biochemical considerations + cell culture plastics, media, conditions, serum, cell cycle, passage number

Things to consider in Cell based assays

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What are you detecting, how would you measure it,does an assay already exist

Basic assay questions

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Adapting an assay

changes in temp, pH buffer, cofactors, co enzymes

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Miniturization

adapting and assay to a smaller volume

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Cofactors

helper molecules, organic/inorganic, don’t bind

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Coenzymes

organic, bind to active site. substrate recruitment

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direct, indirect, colorimetric, fluorescent

Four types of assays

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89

Direct Assay

activity results in a change that can be measured immediately

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Indirect Assay

activity that causes a change that can only be measured through a secondary reaction

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Colorimetric Assay

a change in color is measured, requires a spectrophotometer

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Fluorescent Assay

emitted light is measured, associated with ATP hydrolysis, requires fluorimeter

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Directly qualify a compounds without a standard curve

What do extinction coefficients tell you?

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94

When relating concentration of indicator to another compound

When is a standard curve still necessary?

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SE = SD/SQRT(n)

equation for standard error

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high standard deviation

data near mean

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Low standard deviation

data more spread out

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Standard error

how different a measure sample is from overall population

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Biological repetition

How do you think you can prove your technical measurement is valid? (hint: additional matching experiments)

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100

UV & Visible Spectrophotometers, Fluorimeters, HPLC, Gas chromatography, Gel Electrophoresis, Microscopy

Common equipment used. for assays

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