Molec Final Study Guide
What three substances are needed for electrophoresis?
(1) a power supply (source of electrons) (2) a plastic gel box or tank (3) a fluid of water and ions which partially fills the gel box
How does the TBE buffer stabilize the reaction during electrophoresis?
1) it provides the necessary ions for a current to easily travel through water 2) EDTA with in the TBE buffer absorbs divalent cations which are necessary cofactors for DNAses.3)counteracts the pH changes that occur with the electrolysis of water allowing the electrophoresis to continue for an extended period of time.
Transformation efficiency is expressed as: [the number of transformants]/ [1μg of DNA] What is the transformation efficiency if one had 125 colonies when plating 0.2μg of DNA?
125/.2
What is the significance of the 221bp band in this lab? Can they taste PTC?
221 bp indicates that you are a homozygous recessive tt, or non-taster. You have single band in the same place as the uncut control ( - - ).
How large is the piece of DNA?
440 BP
Which portion of the mitochondrial DNA was amplified in this Lab?
440 BP region of control region.
If a restriction enzyme cuts a piece of linear DNA in 5 locations how many fragments are formed? What if it cuts in 6 locations? What about 3 locations?
5 locations = 6, 6 locations =7, 3 locations= 4
A restriction enzyme cuts a 10000bp piece of DNA in the following locations: a. 500bp, 1300bp, 3000bp, 8500bp, 9400bp. b. What size DNA fragments are created from this digest?
500bp, 800bp, 1700bp, 5500bp, 900bp, 600bp
The complete PCR reaction mix was added to the thermocycler (PCR machine) and cycled through three temperatures, 94°C, ~64°C, and 72°C. Explain what is happening to the DNA at each of these temperatures.
94:denatures the double stranded DNA into single strands. 64:allows the primers to to form hydrogen bonds and anneal with the specific regions of the template DNA. 72:It's for the taq polymerase to assemble the free dNTPs into complementary DNA strands that begin with each primer.
What happens during the 42° heat shock?
A temperature imbalance causes a current to flow that sweeps plasmid DNA into the cell.
What is the role of the promoter found next to the GFP gene?
ARA promoter enables the production and expression of the GFP gene (Green Fluorescent Protein), if it is present in an environment of bacteria.
What feature of a plasmid allows for easy screening of successful transformants?
Antibiotic resistance and GFP gene makes plasmid glow in ultraviolet light.
Restriction Enzymes are isolated from what type of organism?
Bacterial Cells.
Why is there less bacteria growing on the LB + amp plate than there is on the LB plate?
Because ampicillin kills plasmid DNA.
What is the purpose of adding Calcium Chloride solution to the E.coli cells?
Calcium Chloride neutralizes the repulsive force within adhesion zones that would repel traveling plasmids (stabilizes phospholipids).
What is happening at each electrode of the electrophoresis apparatus? (Positive - anode; Negative - cathode)
Cathode(-): 4 electrons- + 4H20-> 2 H2(gas) +4OH- Anode(+)4 H20 --> O2 (gas) + 4H+ +2H20 + 4electrons-
What is happening at each electrode of the electrophoresis apparatus? (Positive - anode; Negative - cathode)
Cathode(-): H2O + O2 (gas) + 4 H+ ‡ 2 H2O + 4 electrons- Anode(+)4 electrons- + 4 H2O ‡ 2 H2 (gas) + 4 OH-
Once the cells are lysed, how are the proteins separated from the DNA?
Centrifugation. This causes two layers to appear: a bottom layer of protein and n upper layer, the supernatant, which contains the DNA.
What do the color changes of phenol red indicate, specifically? At which end of the electrophoresis apparatus did each of these color changes occur?
Changes in color indicate changes in pH levels (i.e. red- acidic yellow-basic).
Why is the DNA loaded in to the negative (Cathode) end for gel electrophoresis?
DNA is added to the negative side so that they can migrate from the negatively charged side of the gel box, to the positive side, creating energy difference which establishes an electric field through which the ions in the gel box fluid migrate. DNA is also negatively charged.
What cation co-factor do DNAses need to break down DNA?
Divalent cations such as Mg2+
What chemical inhibits DNAse by binding its cofactors?
EDTA (Ethylene Diamine Tetraacetic Acid) prevents the destruction of DNA by binding divalent cations.
What is a SNP? Give an example.
Each variable position is termed a single nucleotide polymorphism (SNP) An example: haplotype, correlates most strongly with tasting ability.
What is used to precipitate the DNA from the supernatant?
Ethanol causes the precipitation of DNA as it excluded hydrophilic molecules. DNA cannot dissolve in alcohol.
Explain why we digested our PCR products with the restriction enzyme HaeIII.
HaeIII, whose recognition sequence includes one of the SNPs, and the samples are analyzed by gel electrophoresis. It will successfully cut the 221bp product into two pieces, 44bp and 177bp fragments.
What is the significance of the 177bp band in this lab? Can they taste PTC?
Indicates if you are homozygous dominant. You can taste the PTC.
As mitochondria are ubiquitous in eukaryotic cells, how can the comparison of mtDNA sequences be used to determine the evolutionary relationship between individuals and organisms?
Individuals that are closely related have little to no differences between their mtDNA sequences. This should be especially true for siblings with the same mother as the mitochondria are exclusively maternally inherited. Comparisons between ancestral humans and students revealed variable results depending upon the sequence of each individual student.
When dispensing the contents of a pipette into a microtube, where is the best location to deposit it? Bottom, Top, Side, into already present liquid?
Into the already present liquid.
If a green glowing bacterial colony is grown on a plate lacking arabinose, what color would you expect it to appear?
Its original color. It will not glow green under an ultraviolet light.
How are the fragments of DNA separated at the end of electrophoresis? (Where are larger DNA fragments located relative to smaller DNA fragments)
Larger DNA fragments are closer to the negatively charged side, and smaller to the positively charged side. This is because smaller molecules are able to move through the gel quicker.
Who did your inherit your mitochondrial DNA from?
Mother
If no template was added to the reaction, was kinds of products would be expected from the reaction?
No products because the template is vital. The PCR needs something to adhere to.
The first step in the isolation of DNA for the PCR labs was to rinse out mouths out with a saltwater solution. What were we trying to extract from out mouths?
Our squamous cheek cells(Mitochondrial DNA).
What was the purpose of phenol red in the "exploring electrophoresis" lab? Was this necessary when actually electrophoresing DNA?
Phenol red was used as an indicator for the pH changes occurring in the electrophoresis box. Phenol red isn't necessary for electrophoresis and is merely used a an agent to visualize changes that occurred in the apparatus.
Why are rapidly growing bacterial cells needed for heat shock transformation?
Rapidly growing bacterial cells have regions in their cell membrane that are porous to DNA (adhesion zones) that are large enough to have plasmids travel through them.
What chemical is used to dissolve the cell membrane?
SDS (Sodium Dodecyl Sulfate). This detergent like substance dissolves lipid cell membranes and denatures proteins.
If no TBE buffer was added to the apparatus, how long would the reaction continue, a longer or shorter duration that if there was buffer present?
Shorter period of time because the reaction would grow increasingly unstable.
What was the final substance we added to the PCR reaction mix? What is its purpose?
Supernatant from DNA extraction. Its purpose is to act as the template.
What are the three components of the "PCR Bead?"
Taq polymerase, dNTP's and MgCl2 (a salt magnesium chloride).
Explain why each of the components of the PCR bead is needed in a PCR reaction.
Taq polymerase:A type of polymerase that is resistant to denaturation (breaking down) at high temperatures. It is commonly used to replicate DNA during Polymerase Chain Reactions (PCR). dNTP:building blocks of new DNA strand. MgCl2:the role is similar to that of a catalyst in that the magnesium is not actually used in the reaction but it can not function without its presence.
Explain how the presence of arabinose affects the expression of the GFP gene.
The ARA promoter is activated in the presence of sugar arabinose and in the environment of the bacteria it this enables the expression of the GFP gene and the production of the GFP protein within the cell.
Why must the CaCl2/ E.coli mixture be incubated on ice?
The cold temperatures (1) congeals (solidifies) the lipid membrane (2) stabilizes the phospholipids (3) makes the negative charge easier to shield.
What is the observed relationship between the number of fragments created and the number of times a restriction enzyme cuts linear DNA?
The number and sizes of fragments created depend on the how many times each restriction enzyme's recognition sequence appears in the sequence of Lambda.
Was the "pink stuff" identical for each of the three labs that we did? Explain.
The primer mix was different. These amplifications resulted in amplification of three different regions of our template DNA.
If two strangers had no differences between their mtDNA sequences, what would this mean?
They are closely related.
How could somebody have the 221bp band and the 177bp band as their genotype? Can they taste PTC? Explain how this "tasting" is different from the individuals in numbers 52 and 53.
They can have the two bands because they will be heterozygous for tasting and weakly taste the paper.
If two brothers had 15 differences between their mtDNA sequences, what would this mean?
They have different moms.
Why was this saltwater solution spun in a centrifuge?
To harvest our squamous cheek cells to create a template DNA for the PCR reaction.
The next step was to resuspend the pellet in a Chelex solution and boil it. What was the purpose of the Chelex? What was the purpose of the boiling step?
To remove trace amounts of metal ions and boiled so the cells were then lysed (BROKEN DOWN) in the presence of Chelex beads.
Order the DNA fragments from #27 as they would appear after gel electrophoresis from those closest to the well to those that would travel the farthest from the well.
WELL: 5500bp, 1700bp, 900bp, 800bp, 600bp, 500bp
We then spun the boiled solution in a microcentrifuge. Was the substance that we wanted in the supernatant, or the pellet?
We wanted the supernatant.
What would be the correct setting on the pipette to measure the following volumes:
a. Blue - 100-1000 μl pipette: 500μl: 050 250μl: 025 760μl: 076 b. Yellow - 2-20 μl pipette: 2μl: 020 25.5μl: 200 and 055 5μl: 050
At the end of 30 cycles of PCR, about how many times was the DNA region amplified?
amplified the DNA over 1 billion times.
What was the "pink stuff" that was added to the PCR bead?
mtDNA PRIMER mixed with loading dye.