bio lab practical 2
types of chemical reactions
(a) Synthesis reaction: A + B → AB (b) Decomposition reaction: AB → A+B (c) Reversible reaction: A+B ↔ AB (d) Recombination reaction: AB+CD → AC+BD
protein marker
(sometimes also called a protein ladder) is a commercially prepared protein mixture that acts as a standard - the proteins in this mixture have known molecular weights.
how long does each step take
30 seconds to a few minutes.
how many cycles for a complete PCR reaction
30-40
Would you expect an enzyme to exhibit higher enzyme activity at 0 C or 37 C? Explain your reasoning.
37, that is not too hot that it would denature but hot enough to make the enzymes move around more, which means more collisions with more active sites: more activity
terminator
A DNA sequence called a terminator is added downstream of the gene cDNA. The terminator sequence acts as a stop signal for RNA polymerase, thus stopping transcription of the gene when in the recipient cells
how does a cell use enzymes
A cell can control where and when a chemical reaction takes place by controlling where, when, and how much of a specific enzyme is made
two common example of plant GMOs
Agriculturally important plants like soybeans and alfalfa have been genetically modified with a bacteria gene that makes them resistant to Roundup, an herbicide used to kill weeds and grasses. When fields of these "Roundup Ready" crops are sprayed with the herbicide, nutrient-robbing weeds will die, but the crops are unaffected. The European corn borer is a common corn pest. Some corn plants have been modified with a gene from the bacterium Bacillus thuringiensis. This gene causes the cells of a corn plant to produce a protein normally made by the bacteria and acts as an endotoxin. When a corn borer larva eats part of a "Bt" corn plant, it consumes the endotoxin made by the cells and dies.
function/what would happen if it was gone buffer
Buffer: The buffer ensures that the PCR reaction will occur at the perfect conditions for it with regards to salt concentration and pH levels. It also has Mg2+, which is a cofactor for DNA polymerase. Since the buffer helps the DNA polymerase function, losing the buffer would affect the same step that losing the DNA polymerase would. Without the buffer the DNA polymerase wouldn't work, so the extension step would be affected., the DNA polymerase would not be in good shape to bind to the annealed primers, read the single stranded template DNA, and copy the target sequence.
common promoter
CaMV 35S
DNA nucleoties
DNA polymerase will link them together to make the copies of the target sequence
three temp steps for PCR cycle
Denaturing annealing extension
how does very high temp affect enzyme activity
For many enzymes, very high temperatures can break some or all of the chemical bonds holding together their three-dimensional shapes, causing them to partially or fully denature. A change in overall shape will likely alter the shape of an enzyme's active site, thus inhibiting enzyme-substrate binding.
what are "G Boil", "G Nat", "B Boil", "B Nat", "IG Boil" and "IB Boil"
G means the purified GFP B means purified BFP I means impure, still a mixture Nat means with its natural shape, not denatured boil means boiled so the natural shape will be lost
GMM
GMO Master Mix, which contains the same basic PCR components and primers to target the CaMV 35S promoter or the NOS terminator present only in genetically modified plant DNA. The mix is colored with a red dye for easier identification.
why glycerol
Glycerol is a dense molecule that will weigh down the protein sample so that it stays where you load it into the gel.
why do you not want a native protein in electrophoresis and what do yo do about it
In electrophoresis, charges and native shapes can interfere with a protein's ability to migrate solely by its size. To avoid these possible inconsistencies, native proteins are treated with a protein denaturing solution before they are placed in a gel
ph and enzyme activity
Most enzymes have an optimum pH where their activity is greatest. For example, the optimum pH of enzymes in human blood is around 7.4. For these enzymes, an environment that is too acidic or basic might cause them to denature. Some enzymes, however, are designed to work at extreme pHs. For example, the optimum pH of enzymes in the human stomach is around 2. These enzymes would have less or no activity at pHs greater than 2. the wrong pH denatures the enzyme
common terminator
NOS
PCR copies what vs DNA
PCR copies a specific target DNA sequence instead of copying all of the DNA in a cell.
PCR uses what for the steps vs DNA
PCR uses different temperatures instead of different proteins to carry out most steps of DNA replication. However, DNA polymerase is still used in PCR to copy the DNA sequence of interest.
PMM
Plant Master Mix, which contains PCR components (buffer, nucleotides, DNA polymerase) and primers to target the chloroplast sequence present in all plant DNA. The mix is colored with a green dye for easier identification.
what kind of gels are used for electrophoresis
Polyacrylamide gels, made from cross-linked acrylamide molecules, are frequently used for electrophoresis because of their ability to separate molecules like proteins or DNA.
function/what would happen if it was gone primer
Primers go around the target sequence (the sequence that is about to be copied). It is a short sequence of single stranded DNA. Without the primer, the annealing step could not happen, there would be nothing to form the base pairs.
how does the active site work
The active site of an enzyme is a specific part of an enzyme's shape that comes in contact with the substrate(s). The interaction between enzyme active site and substrate(s) is similar to a lock and key - their shapes must be compatible
column matrix
The column matrix consists of small beads that contain charges or pores along their surface. The beads are suspended in a clear buffer
what do protein denaturing solutions do
The denaturing solution dismantles protein shapes and ensures that all proteins have a net negative charge.
denaturing
The reaction is heated to a high temperature to break apart the base pairs holding together the nucleotide strands of the double-stranded template DNA. 94C.
function/what would happen if it was gone template DNA
The starting point of the target sequence. The annealing step would be affected by the absence of template DNA, without the template DNA the primers would have nothing to form base pairs with.
tracking dye why
This is a blue, negatively charged dye of low molecular weight that will migrate quickly through the gel during electrophoresis. Since its migration rate will be faster than the colorless proteins in your samples, the tracking dye will help you determine when to stop the electrophoresis.
extension step
This is the step where the target sequence is actually copied. The temperature is raised to 72oC, which is ideal for the DNA polymerase to bind to the annealed primers, "read" the single-stranded template DNA, and copy the target sequence by adding the DNA nucleotides supplied in the reaction.
function/what would happen if it was gone DNA polymerase
This will bind to and extend the primers so they can make a copy of the DNA sequence that is targeted. Without DNA polymerase, the extension step would be directly affected, because the polymerase wouldn't be there to bind to the annealed primers, read the single stranded template DNA, and copy the target sequence.
promoter
a DNA sequence called a promoter is added upstream of the gene, so that transcription will be initiated properly by RNA polymerase
PCR
a molecular biology technique used to clone, or make identical copies of, a specific DNA sequence
native protein
a protein that retains its natural shape
what is GFP shape
a protein with a 238 amino acid structure. it has a cylindrical shape which protects the flurophore, which creates florescence. A GFP molecule is actually two GFP proteins bound together. two cylinders bound together
coomassie blue
a stain that will color all proteins after electrophoresis
chromatography
a technique that separates molecules that are in a mixture
how Column chromatography works
a tube is filled with column matrix. the picture is put in the top, along with additional buffer to help it flow through. the molecules separate as they proceed through the matrix. they separated molecules elute from the bottom and are collected in small volumes called fractions
what happens when an enzyme is saturated
all available enzyme active sites are occupied and working at maximum efficiency. At enzyme saturation, any additional increase in substrate concentration will not increase enzyme activity.
which template DNA will have a positive result for the PCR reaction with Plant Master Mix
all of them, because they all are plants with plant DNA
enzyme is
an organic molecule (usually a protein) that speeds up a specific chemical reaction
GMO
an organism with DNA that has been deliberately altered, often by adding DNA from another organism
substrate concentration and enzyme activity
as substrate concentration increases enzyme activity does too, until it reaches saturation (more collisions, more active sites binding, more activity)
how does TLC separate the molecules by charge
as the mobile phase moves through, the pigment molecules that are charged will be more attracted to the silica coating, slowing their migration. Pigment molecules that are less charged or completely uncharged will be more attracted to the mobile phase, making their migration faster. Thus, charged pigments will appear as horizontal bands closer to the origin, while less charged or uncharged pigments will appear as bands farther away from the origin.
DNA polymerase
binds to the primers and extends them to copy the target DNA sequence
thermocycler
contains a metal block that holds the tubes and can be quickly heated and cooled to the temperatures required for PCR
denaturing solution is made of
denaturing reagents, glycerol, tracking dye
Rf formula
distance from origin to band/distance from origin to solvent front
what do enzymes need
energy: ATP
which template dina will have positive results for the GMO master mix
for sure GMO, and the test food if it is a GMO food
The U.S. Food and Drug Administration requires that any food labeled as "organic" must be
free of GMOs
what catalyzes 2 glucose + O2 + 2 H2O -> 2 gluconic acid + 2 H2O2
glucose oxidase
objections to GMO food
herbicide resistant superweeds could show up because of cross pollination between GMO plants and weeds Pests could develop resistance to toxins in genetically modified plants allergies from GMO foods could have unforeseen impacts on public health
When you have an infection, you might have a fever. Why does raising your body temperature help slow the spread of infectious bacteria?
higher temperatures denature the enzymes in the infectious bacteria
why would the pigment bands be too faint
if you didn't apply enough, do several layers
PCR occurs where vs DNA
in a test tube rather than a living cell
electrophoresis
is the process of placing molecules in a porous matrix, such as a gel, and applying an electrical current to move them through the matrix. This technique can be used to separate molecules according to their size, shape and charge.
what does an enzyme work as
it acts as a catalyst, speeds up a specific chemical reaction
how does an enzyme catalyze a reaction
it binds to the substrates with its active site
BFP structure
it is a protein dimer like GFP
what is in the mobile phase
it is a solvent picture of petroleum ether, acetone, and chloroform
how to find the absorption maxima
it is the actual peak like /\
BFP origin
it was made by altering the DNA of a GFP gene
what will elute from sephadex first? why?
large proteins, they go around the beads which is faster than the small ones that go through
where will the large and small proteins be after electrophoresis
larger ones will be slower (close to well end), smaller ones faster(close to bottom )
which pigments are most attracted to the mobile phase
least charged ones, they will end up closest to solvent front
A cut avocado turns brown because of the activity of an enzyme called catacholase. One way to prevent browning is to add lime juice to the cut avocado. Why does the lime juice prevent avocado browning?
lime juice changes the ph. that denatures the catacholase, which will stop the browning
what denaturing reagents do we use and why
mercaptoethanol, which breaks disulfide bonds SDS which breaks weaker bonds and negatively charges proteins
would you expect a protein picture to appear as a single band or multiple
multiple, a mixture means different proteins at different weights, which means they will separate
why would the pigment bands appear uneven
oils from hands interfere with the migration molecules if the silica was scraped all the way off when you applied the pigment picture that could mess it up the pigment wasn't applied in a straight line, or you applied it all the way to the sides you didn't close the chamber all the way
sephadex beads
porous polysaccharide bases beads. they are covered in pores. too small to see with the naked eye
primers in PCRvs DNA
primers in PCR are made of DNA instead of RNA
Annealing step
primers will form base pairs with each single-stranded template DNA. . Your annealing step will occur at 59 C.
what goes through sephadex beads faster
proteins that are small enough to fit into the bead pores will move into and out of the pores and around the beads. Proteins that are too large for the bead pores can only travel around the beads. Since it is faster to go around the beads than it is to go into bead pores and around the beads, larger proteins will elute from the column faster than smaller ones.
buffer
provides ideal pH and salt concentration for PCR, including Mg+ which is an important cofactor for the function of DNA polymerase
substrates
reactant molecules
5 components of PCR
sample dna (template DNA) primers DNA polymerase DNA nucleotides buffer
thin layer chromatography is used to
separate pigments according to charge
what did we use polyacrylamide gel to do
separate proteins by size
GFP size exclusion column chromatography consists of
sephadex beads, buffer
bottom end of gel is
the anode end, positively charged
what is between promoter and terminator
the cloned gene cDNA
how will the not denatured and not heated protein samples become negated.
the gel is submerged in an electrophoresis buffer that contains SDS. Glycerol will be added to these native samples before they are loaded to keep them in the gel.
where does GFP come from
the gene for producing it has been isolated from jellyfish DNA.
how will the end result of electrophoresis be
the large ones won't be able to get through. they will form the bands closest to the well end: the cathode. they are negatively charged from the SDS so they will want to move to the bottom and the positively charged anode but the cross linked acrylamide will get in the way
solvent front
the leading edge of the solvent
Vmax
the maximum activity at enzyme saturation. the enzymes maximum velocity
the darker the band
the more protein molecules in that band
which one is the one most attracted to the stationary phase
the most charged one, the one that will be closest to the origin
stationary phase is
the part that is coated with charged silica.
what happens if the column runs dry of buffer
the pores in the beads close, that means there will be no difference in the pathways of small vs large proteins, and the column is not functioning
how/ why will the proteins separate in electrophoresis
the pores in the cross linked acrylamide will hinder the migration of the proteins to different degrees. the larger ones will migrate slower than smaller ones.
Template DNA is
the source of target sequence
what happens if the pigment mixture dissolves into the solvent
there will be no pigment bands on the TLC plate, and the solvent in the bottom will be brightly colored. to avoid this make sure the pigment mixture is applied above the level of the solvent.
function/what would happen if it was gone DNA nucleotides
these will be linked together by the DNA polymerase to make a new copy of the target sequence. The extension step would be affected without the DNA nucleotides, because without them the DNA polymerase could not add anything when they copy the target sequence.
what are phenol and 4 aminoantipyrine used for
they measure the amount of H2O2 produced by the reaction, they react with H2O2 to become pink. then you can measure the absorbance of the pink dye, which will be the measurement of the glucose oxidase's activity
support arguments for GMO food
they say that GMO crops are more environmentally friernfsly because they dont require as many toxic herbicides and pesticides they also could be made to grow in conditions that aren't suitable for normal crops
why are protein samples heated
to eliminate any remaining interactions holding together a proteins native shape
GMOs are sometimes called
transgenic organisms
warm vs cool temperatures and enzymes
warm temps let the enzymes move faster and have higher activity, low temps make it more slower so they have low activity
which side is anode/cathode for electrophoresis
well end: negatively charged: cathode bottom: positively charged: anode
mobile phase
what the paper with the stationary phase on it is dipped in. it moves up the paper, moving the pigments
origin is
where you apply the thing that is going to be separated
well end is
where you put in the samples, it is the negatively charged end, it is the top
primers are
which are short sequences (around 20 nucleotides long) of single-stranded DNA that are manufactured so that they will flank the target sequence that is to be copied. In a living cell, primers would be single-stranded RNA.
what if the TLC pigment bands are broad and overlap
you didn't let the pigment dry between layers
what is BSA used for
you know the concentrations so you use it to set up a standard curve. of protein concentration vs Absorbance. then you find the absorbance of the purified GFP and BFP, and insert them into the curve to find the absorbance