Lab Test 2 Flashcards
Can you explain some purposes of generating genetically modified crops plants?
- to encourage specific traits such as…
high yield, undesirable to pests, delayed fruit ripening, reistant to drought
Can you briefly explain what is a GMO?
Farmers have been genetically modifying crops for centuries and crop breeding to encourage specific traits, such as high yield, is still an important part of agriculture today. However, there is now the option to place genes for selected traits directly into crop plants. These genes do not have to originate from the same plant species—in fact, they do not have to come from plants at all. GMO is the genetic modification of a plant in order for traits to be amplified or introduced into an organism.
What is the general purpose of PCR?
PCR is DNA replication in a test tube. PCR allows you to amplify specific sections of DNA and make millions of copies of the target sequence.
To prepare a DNA sample for PCR, do you know which reagents are required and what is their
respective function(s):
o Template DNA (what is this)?
This is the DNA molecule containing the segment of DNA you wish to amplify.
o How many primers/oligonucleotides per target DNA sequence to be amplified?
Small pieces of single stranded DNA (typically 18 to 25 bases long) that bind to complementary regions on opposite strands of the template. The primers serve as replication start points. Two primers are used to amplify one DNA segment.
o DNA polymerase
The enzyme used to replicate DNA. It adds bases onto the 3’ ends of the annealed primers. For use in PCR, the DNA polymerase should be stable at high temperatures. In PCR reactions, the enzyme Taq DNA polymerase isolated from bacteria that thrive in the hot springs is used.
o MgCl2
Provides magnesium ion (Mg2+) to the reaction. Mg2+ is a cofactor for DNA polymerase. Without Mg2+, DNA polymerase will not function.
o dNTPS
(deoxynucleoside triphosphates). dNTPs is the term used to refer to the four deoxyribonucleotides: dATP, dCTP, dGTP and dTTP needed to replicate the template DNA. The nucleotides are added by the Taq DNA polymerase enzyme onto the 3’ ends of the annealed primers.
-Buffers
Maintains the pH of the reaction at a level where the DNA polymerase is most active.
What is the sequence of the main steps in PCR in the thermal cycler? At what temperatures are each
of these steps performed? Why?
o Denaturation
The first level is at a high temperature and is used to break the hydrogen bonds between the complementary bases that hold the two strands of the template together.
-95°C water bath
o Annealing
The reaction is then taken down to a lower temperature at which point the two PCR primers can anneal to the template strands.
o Extension
the reaction is brought up to an intermediate temperature at which the DNA polymerase adds nucleotides onto the ends of the annealed primers.
What is the relationship between the number of cycles a PCR has and the amount of DNA produced?
The steps of denaturation, annealing, and extension, make up one complete cycle. Making enough copies of DNA for analysis by gel electrophoresis typically requires from 25 to 40 cycles.
During the reaction, each new DNA fragment made in one PCR cycle can serve as a template in the next. This results in a doubling of the amount of amplified PCR product with each cycle. Mathematicians call this exponential amplification and describe the rate at which new products accumulate by the expression 2^n, where n is the number of cycles. If a PCR amplification is carried through 40 cycles, for example, the DNA would be amplified by 2^40.
In experiment 5A, what two master mixes used?
PMM (455bp) and GMM (255bp)
What were the positive controls in experiment 5A? The negative control?
+ve GMM (A positive control is a group in an experiment that receives a treatment with a known result, and therefore should show a particular change during the experiment)
-ve: Non-GMO food (oats)
Why were the food samples mechanically ground as the first step of this experiment?
The food samples were ground up to release the DNA that we wanted to extract
Why was it important to keep all the DNA samples on ice during the last steps of DNA extraction
(before placing them into the thermal cycler for PCR)?
to preserve the integrity of the samples and reagents and prevent thermal degradation of the proteins and enzymes being studied. Heat denatures the enzymes causing non-specific annealing and unwanted by-products.
Is gel electrophoresis used to separate only DNA bands? What other biomolecule have you used this technique to separate in our BZE labs?
We have used gel electrophoresis to separate proteins
How were the DNA bands in the lanes separated? By size? Shape? Structure?
Molecular weight
What is the unit for DNA size?
bp
Why was a DNA ladder loaded into a lane?
so we can determine around what size our DNA samples are when comparing it to known molecular weights
Why was loading dye (EZ-VisionTM) added to each DNA sample before it was loaded into a gel well?
So we can view the solution as it travels down the wells
Why does DNA migrate toward the positive electrode?
Due to its negative charge
Why was it possible to see the separated DNA bands in the gel?
The molecules can only pass through so far in the gel due to how it they arent all small enough to travel all the way towards the end
In the gel from this experiment, did a lane that didn’t have the GM promoter sequence
necessary mean that the food was not from a GMO?
No. It just means that it was not amplified
Are you able to label and give the function of cells and structures of different leaf cross sections;
cuticle, upper epidermis, lower epidermis, palisade mesophyll, spongy mesophyll, vascular tissue,
xylem, phloem, stomata, guard cells?
cuticle,
outside of leaf, usually waxy
upper epidermis,
lower epidermis,
palisade mesophyll,
the parenchyma (functional) tissue that lies between the upper and lower epidermis, excluding vascular tissues. the parenchyma (functional) tissue that lies between the upper and lower epidermis, excluding vascular tissues.
spongy mesophyll,
the parenchyma (functional) tissue that lies between the upper and lower epidermis, excluding vascular tissues. spongy mesophyll containing irregularly shaped cells beneath the palisade layer and having many intercellular spaces.
vascular tissue,
of vascular bundles, recognizable on the whole leaf as veins. (Made of xylem and phloem)
xylem, :
the xylem (upper portion, thick-walled cells, stained red)
phloem,
the phloem (lower portion, small, thin-walled cells, stained green).
stomata,
The stomata (singular stoma) are the systems made up of pores and guard cells through which gas exchange (CO2 in, O2 out) occurs and are usually located on the lower surface of the leaf.
guard cells,
); the guard cells, pairs of very small cells bordering stomata, can close or partially close to prevent excessive water loss
If you know the structural differences between different types of plant leaves, can you explain how
they reflect the adaptation of those plants to different environments with respect to: water conservation, gas exchanges & light absorption?
WATER
Water loss through stomata is replaced by upward diffusion of water from lower parts of the plant. This whole process is referred to as transpiration, a process of great advantage to plants since water moving up from the soil is rich in minerals. However, if the water lost in transpiration is greater than that replenished by roots (as may occur in dry conditions); the guard cells, pairs of very small cells bordering stomata, can close or partially close to prevent excessive water loss
AIR
stomata opening and closing depending on temperature and humidity
LIGHT ABSOPRTION
Different pigments to optimize light capture
Are you able to identify a mesophyte, xerophyte or hydrophyte leaf in a microscopic cross section by
characteristics and identify their components
hydrophyte: lots of air pockets in the spongy mesophyll
xerophyte: circles of space near lower epidermis
mesophyte: “uniform” look
Do you understand the structure and function of stomata:
o When are guard cells turgid? Flaccid? How are these states related to opening and closing
the pore?
o Why do guard cell have chloroplasts?
o How do the proton pumps in guard cells affect osmosis and pore-opening and closing?
o When are guard cells turgid? Flaccid? How are these states related to opening and closing
the pore?
When guard cells are turgid, they become kidney-shaped (in dicotyledon plants) causing the stoma to open; when guard cells are flaccid, they take on a semi-circular shape causing the stoma to close. A guard cell’s shape is
determined by its water content which, in turn, is controlled by its intracellular osmolarity. When guard cell osmolarity becomes greater than the extracellular fluid, water enters the cell via osmosis causing turgidity and stomatal opening. Conversely, when intracellular osmolarity becomes lesser than the extracellular fluid, water leaves the cell causing flaccidity and stomatal closure.
o Why do guard cell have chloroplasts?
They control the stomatal pore, which serves as a channel for exchange of gas by balancing between CO2 uptake for photosynthesis and water loss through transpiration. As a result, chloroplasts in the guard cells have become potential tool for manipulation toward improvement of plant productivity through photosynthesis.
o How do the proton pumps in guard cells affect osmosis and pore-opening and closing?
The absorption of water is inhibited when guard cells block the stomata’s opening
- Do you understand the difference between absorption & reflection of light, & that the light that is
absorbed “powers” photosynthesis
light from e- excitation (caused by the absorption of photons) is harvested for photosytensis via PSII and PSI. Additionally, all light that is not absorbed is reflected, emitting a plant’s green hue.
Do you understand how paper chromatography separates the pigments (what other molecules can
chromatography separate?
the pigments are seperated based on the molelcules’ polarity. double bonds and oxygen molecules impact the polairty of the lipid.
Chromatography can be used to separate the components in a mixture of biomolecules (such as amino acids or lipids). The molecules are separated based on their solubility in a particular solvent vs. their attraction to a particular solid material.
