Lab Final - PCR, Electrophoresis, and Spectrophotometer

antibiotic resistance

genetic trait encoded in DNA that provides bacteria resistance from a certain antibiotic

vertical transmission

resistant bacteria divide and pass the resistance directly to the daughter cells

horizontal transmission

resistance genes are transferred between bacteria or acquired from free DNA in the environment

1. plasmids

2. transposable elements

two types of genetic material involved in horizontal transmission

plasmids

smal, circular DNA molecules that exist independently of the bacterial chromosomes and often carry resistant genes

transposable elements

DNA sequences located on chromosomes that can move from one location to another, jumping between chromosomes or onto plasmids in different bacteria

tetracycline

antibiotics that bind to the 16S rRNA in bacteria; this prevents tRNA from binding, which stops translation, protein production, and division of cell, eventually killing the bacteria

efflux pumps

proteins in the cell membrane the expel tetracycline out of the cell faster than it enters, therefore providing resistance to tetracycline

tetB gene

tetracycline resistant gene that codes for a pump that removes tetracycline from the cell

tetM gene

tetracycline resistant gene that codes for a protein that attaches to the 16S rRNA; this way, the tetracycline has no where to bind to, rendering it useless

environmental DNA or eDNA

DNA that is collected from the environment and may come various organisms such as bacteria or fungi. In the PCR lab, eDNA was collected from the soil

bead-beating

process where soil is combined with lysis buffer and small beads in a screw-top microcentrifuge tube; then it is shaken using vortexer. The force breaks open microbial cells and releases DNA

polymerase chain reaction (PCR)

a method used to amplify or increase the amount of a specific sequence of DNA

template DNA

DNA that is going to be amplified by the PCR

oligonucleotide primers

lab-made DNA fragments that are complementary to the DNA template allowing the primers to bind to specific locations on the template. These primers will later tell the taq polymerase which section of the DNA to amplify

deoxyribonucleoside triphosphates (dNTPs)

nucleotides used to build the amplified DNA

Taq polymerase

a DNA polymerase that adds dNTPS and creates new, amplified DNA

magnesium ions

cofactors that are needed for taq polymerase activity

salt buffer

needed in PCR to maintain the optimal ionic conditions and pH

1. denaturation

2. primer annealing

3. primer extension

three steps done in one PCR cycle

denatruation

everything is heated to 94 degrees. the heat inactivates natural DNA polymerase and separates the DNA into two single strands

primer annealing

temperature lowered to 60 degrees. primers locate and bind to complementary sequences on the single stranded DNA templates

primer extension

heated to 72 degrees. taq polymerase synthesizes new DNA by adding dNTPs to primer and producing complementary DNA strands and double the amount of template DNA

thermocycler

machine where PCR is done; it automates the timing and heating/cooling of each cycle

temperature cycling or thermal cycling

process of rapid heating and cooling of the PCR tubes

agarose gel electrophoresis

technique that is used to separate high molecular weight molecules, such as proteins and nucleic acid

1. size

2. electrical charge

3. shape

how are the molecules in gel electrophoresis separated by?

agarose gel

molecular sieve that separates the different sized molecules in electrophoresis

the lower the concentration of agarose, the larger the pore size. Larger pores result in larger molecules being able to move through the gel

how does the concentration of the gel affect how the molecules are separated?

anode

positive node of the electrophoresis chamber

cathode

negative node of the electrophoresis chamber

DNA is negative so it will travel toward the anode

during electrophoresis, in node does the DNA travel towards

molecular mass ruler

mixture of DNA of known sizes that run in one lane of agarose gel alongside the samples. serves as a reference to estimate size of unknown DNA fragment

Since 16s is in all bacteria, if it was amplified, we know that we successfully extracted bacterial DNA from the soil; This is a positive control for bacterial DNA

for the tube with 16S primers, what does its amplification tell us?

no bands should appear for these tubes, because there was no DNA template to amplify in the first place. By doing this test, we confirm that the PCR reagents were contaminated with any DNA for prior testing

two of the tubes had negative controls added instead of the eDNA. what are the expected results for electrophoresis?

amplification should occur because there was DNA present in the tubes. This test confirms that the PCR conditions and primers are capable of detecting any resistance genes

two of the tubes had positive controls added instead of eDNA. These positive controls are DNA segments that are known to have tetB and tetM genes. what are the expected results for electrophoresis.

primary producers

organisms that capture energy from sun through photosynthesis

trophic levels

the position an organism occupies in a food chain or food web based on how it obtains energy

primary consumers

second trophic level where organisms eat producers and are one step removed from the original energy source

eutrophication

process in which a body of water becomes enriched with excess nutrients, primarily in nitrogen and phosphorus

1. agricultural runoff

2. sewage discharge

3. fertilizers

4. detergents

what are some ways that eutrophication can occur?

it results in rapid growth and algal bloom. This increased number of algae makes the water opaque and blocks sunlight. When the algae eventually die, decomposers break them down and also a lot of oxygen, depleting. Hypoxia occurs in the water, stressing and/or killing organisms in environment

what happens to primary producers like algae if eutrophication occurs?

primary productivity

the rate at which producers convert solar energy into chemical energy through photosynthesis

chlorella

green algae and primary producer that produces oxygen as a byproduct; it influences oxygen levels in the environment and absorbs nitrogen and phosphorus, creating a bloom

Daphnia

crustaceans that consume Chlorella and respond rapidly to changes in oxygen levels and nutrient availability making them important bioindicators of water quality; they keep algal populations in check

Beer-Lambert Law

states that there is a direct relationship between the absorbance of light and the concentration of the absorbing substance.

A = absorptivity concentration pathlength

A spectrophotometer shows how much light is absorbed. If there is a high amount of light being absorbed, then according to the Beer-Lambert law, there should also be a high concentration of algae.

how does using a spectrophotometer tell us an indirect measurement of the algea

C = absorbance (measured by spectrophotometer) divided by absorptivity

what is the formula for finding concentration, if we know that typically, the pathlength is 1