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BIO 219 Finals Study Set: Questions & Complete Answers
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BIO 219 Finals Study Set: Questions & Complete Answers
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BIO 219 Finals Study Set: Questions & CompleteAnswers
Bacterial Chromosomes Right Ans - Bacterial chromosomes are located,
relatively, centrally in the cell in a region called the nucleoid. Unlike
eukaryotes, bacteria do not have a nucleus and therefore have "naked DNA."
Also, unlike eukaryotes, bacteria have one, contiguous (closed loop) of double
stranded DNA—making it circular in nature
bacterial chromosome Right Ans - Chromosomal DNA (chDNA) is
measured in base pairs (bp) and is often written with a designation prefix of
kilo- (k) or millin (m-) as kbp and mbp, respectively. Take, i.e., A. fischeri has a
genome size of ~4.38 mbp (million base pairs)
Extracting chDNA Right Ans - In order to extract chDNA you will first need
to pellet the cells and resuspend them in specific reagents.
Step 1: Harvest the cells by pelleting by centrifugation
Step 2: Lyse the cells uses a resuspension buffer with reagents designed to
permeabilize the cell membrane, while keeping DNA intact
Step 3: Incorporate reagents that degrade unwanted proteins and RNA,
ensuring mostly DNA gets left behind
Step 4: Apply the cell lysate to a Silica Spin Column, which will enable the DNA
to form a non-covalent bond with the silica resin thus holding it in place while
the rest of the unwanted cell materials pass through the column. A
hydrophobic environment strengthens the bond between DNA and the silica
column
Step 5: Wash multiple times with ethanol to ensure that only DNA is left
behind, still bound to the silica column
Step 6: Rehydrate the silica column, thus restoring a hydrophilic environment
and allowing the DNA to disassociate with the silica column and become
collected for further use
Resuspension Buffer (Buffer ATL) Right Ans - The buffer contains sodium
dodecyl sulphate (SDS) a strong anionic detergent that can solubilize the
membrane proteins. The detergent disrupts the lipid bilayer of gram-negative
cells and brings the proteins into solution as protein-lipid-detergent
complexes. The phospholipids in the membrane are also solubilized by the
detergent. SDS helps release the DNA binding proteins by denaturing them
,and binding both membrane and non-membrane proteins as monomers.
There may be an appearance of small soap bubbles.
Proteinase K Right Ans - Proteinase K is an enzyme that will aid in the
release of nucleic acids while deactivating nucleases (enzymes that degrade
nucleic acids) present. The addition of proteinase K degrades these nucleases
and protects the nucleic acids from nuclease attack. In addition, proteinase K
is stable over a wide pH range and is well suited for use in DNA extraction.
Rnase A Right Ans - Rnase A is an efficacious ribonuclease that is used to
degrade RNA that is present in the sample. It cleaves the 3' side of the
phosphodiester bonds after pyrimidine nucleotides in single stranded RNA.
DNA will remain intact because it does not have the 2'-hydroxyl group
required by Rnase A for forming the cyclic intermediate cleavage product.
Lysis Buffer (Buffer AL) Right Ans - The Lysis buffer contains a chaotropic
salt (guanidium chloride). hydrogen bonding is essential to the secondary
structure of polymers such as DNA, RNA, and proteins, as well as their water
solubility. In vivo, nucleic acids are covered by a hydrate shell consisting of
water molecules that maintain the solubility of DNA in aqueous solutions.
Chaotropic Right Ans - The term chaotropic means chaos forming, which is
a reference to the ability to disrupt the regular hydrogen bond structures in
water.
a chaotropic agent is a molecule in water solution that can disrupt the
hydrogen bonding network between water molecules. ( exerts a chaotropic
activity)
guanidinium chloride Right Ans - When guanidium chloride is added to the
nucleic acis, the hydrate shell and the hydrophobic interactions between
neighboring stacks of base pairs are disrupted. this sets up the conditions for
the DNA to selectively bind to the silica resin in the transfer tube. Chaotropic
salt also further denature residual proteins.
Ethanol (binding) Right Ans - The ethanol will enhance and influence the
binding of nucleic acids to silica by aiding the lysis buffer in creating a more
hydrophobic solution
,Wash buffer 1 (AW1) Right Ans - The AW1 wash has a low amount of
chaotropic salt that binds to and removes the proteins and colored
contaminants
Wash Buffer 2 (AW2) Right Ans - The AW2 contains ethanol to remove the
salts added from AW1.
DNA grade water Right Ans - The DNA grade water free of salts, Dnases,
and Proteases which allows for the re-hydration and renaturing of the DNA,
causing it to lose affinity for the silica. (pH 5.4-7 at 25° C)
Binding to a silica spin column Right Ans - SDS dissociates in the presence
of chaotropic salts in the lysis buffer and forms a cation bridge to bind DNA to
the silica membrane in the spin column
Spectrophotometric Analysis Right Ans - Spec analysis utilizes light. When
light passes through a prism it breaks into each of its wavelengths. These
wavelengths then pass through a thin slit , which is selected for by the
machine.
A detector sits on the other side of the slit, with the sample in between. As
light passes through the sample, specific material will absorb, or pick up, the
light. The remaining amount of light that passes through the sample is picked
up by a detector and is given an absorbance output.
Spec Analysis Example Right Ans - Example: Setting a spectrophotometer
to measure a wavelength of 430nm slects for a blue color and thus will move
the slit to the blue light.
In case of the picture the slit would move downward letting only blue light
pass through.
Spec analysis of chDNA Right Ans - DNA STRONGLY absorbs light in the UV
range (10nm-400nm)
When DNA absorbs the UV, the remaining light (not absorbed by DNA) is
picked up by a detector and can give a direct output to the concentration of
DNA in a given sample
, To do this the Nanodrop(Spectrophotometer) employs the Beer-Lambert law
equation.
Spec Analysis of chDNA pt2 Right Ans - The maximum wavelength of DNA
absorbance is 260nm, again within the UV range. This is due to the
nitrogenous bases of DNA (Adenine, cytosine, guanine, and thymine)
Due to the lack of phosphate backbone single stranded DNA (ssDNA) has a
higher absorbance of this wavelength
The output absorbance is then transferred to the Beer-Lambert law Equation,
where the concentration of DNA is empirically derived.
Beer-Lambert Law Right Ans - A = εcl
A = absorbance value (no units)
ε = extinction coefficient (molar absorptivity) (constant for each substance,
units of M-1 cm-1, in the case of DNA µg/mL.
c = concentration of substance (units of M or µg/mL)
l or (b)= light path length (in cm); all specs in common use have l = 1 cm
The extinction coefficient of dsDNA empirically found. The value is 0.020 per
μg double stranded DNA per mL of solution per cm of light-path, or 0.020 per
μg/mL-cm
Therefore, when plugging this number into the equation to the left you will
end up with units of µg/mL. The NanoDrop, however, will display the units in
ng/uL and will need to be converted.
Beer-Lambert Law, Example Right Ans - Use the corrected absorbance to
calculate the concentration of your chDNA sample with an equation derived
from the
Beer-Lambert Law:
A = εcl
A = absorbance value (no units)
ε = extinction coefficient [0.020 (μg/mL)-1(cm)-1]
c = concentration of substance (units of μg/mL )
l = light path length (in cm); all specs in common use have l = 1 cm
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