Molec Final
the process of Polymerase Chain Reaction (PCR) that was a component of the mtDNA and PTC Labs: 42. The complete PCR reaction mix was added to the thermocycler (PCR machine) and cycled through three temperatures, 94 degrees C, ~64 degrees C, and 72 degrees C. Explain what is happening to the DNA at each of these temperatures.
-94* C : denatures the double stranded DNA into single strands -64* C: allows the primers to form hydrogen bonds and anneal with the specific regions of the template DNA -72* C : the taq polymerase can assemble to the free dNTPs into complementary DNA strands that begin with each primer
the process of Polymerase Chain Reaction (PCR) that was a component of the mtDNA and PTC Labs: 36. What are the three components of the "PCR bead"?
-dNTPs -MgCl2 - Taq polymerase
the process of Polymerase Chain Reaction (PCR) that was a component of the mtDNA and PTC Labs: 37. Explain why each of the components of the PCR bead is needed in a PCR reaction.
-dNTPs: building blocks of new DNA strand -MgCl2: salt necessary for DNA polymerase to properly function - Taq polymerase: DNA polymerase isolated from a thermophilic bacteria that can maintain its native activity despite being heated to almost boiling temperatures. It is resistant to denaturation (breaking down) at high temps.
Electrophoresis Lab and Gel Electrophoresis of DNA: 9. What three substances are needed for electrophoresis?
-power supply (source of electrons) -a plastic gel box or tank -fluid of water and ions which partially fills the gel box
Electrophoresis Lab and Gel Electrophoresis of DNA: 11. How does the TBE buffer stabilize the reaction during the electrophoresis?
-provides the necessary ions for a current to easily travel through water -EDTA with in the TBE buffer absorbs divalent cations which are necessary cofactors for DNAses -counteracts the pH changes that occur with the electrolysis of water allowing the electrophoresis to continue for an extended period of time
Transformation of E. coli Lab: 23. Transformation efficiency is expressed as: [the number of transformants]/ [1 ug of DNA] What is the transformation efficiency if one had 125 colonies when plating 0.2 ug of DNA?
125/0.2 ug = x/1 ug = 625 transformants/ug
Restriction Enzyme Lab: 28. 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?
5-6 6-7 3-4
Restriction Enzyme Lab: 30. A restriction enzyme cuts a 10,000 bp piece of DNA in the following locations: a. 500 bp, 1300 bp, 3000 bp, 8500 bp, 9400 bp. What size DNA fragments are created from this digest?
500 | 800 | 1700 |5500 | 900 | 600 0 500 1300 3000 8500 9400 = 10000
PTC PCR Lab: 50. What is a SNP? Give an example.
A SNP is a single nucleotide polymorphism. They are inherited through generations. An example of one is termed a haplotype, which correlates most strongly with tasting ability.
Transformation of E. coli Lab: 20. What happens during the 42 degrees heat shock?
A temperature imbalance causes a current to flow that sweeps plasmid DNA into the cell
the process of Polymerase Chain Reaction (PCR) that was a component of the mtDNA and PTC Labs: 43. At the end of 30 cycles of PCR, about how many times was the DNA region amplified?
After 30 cycles, we will have amplified the DNA over 1 billion times. 1 cycle-doubles it
Transformation of E. coli Lab: 21. What feature of a plasmid allows for easy screening of successful transformants?
Antibiotic resistance gene is able to show the positive transformants on bacterial media that had antibiotics.
Electrophoresis Lab and Gel Electrophoresis of DNA: 10. What is happening at each electrode of the electrophoresis apparatus? (positive-anode, negative-cathode)
At the negative cathode, electrons enter the gel box from the power supply along the black wire and is negative. @ cathode: 4 electrons- + 4H2O ---> 2H2 (gas) + 4 OH- At the positive anode, electrons leave the box and re enter the power supple along the red wire and is positively charged. @ anode: 4 H2O ---> O2 (gas) + 4 H+ + 2H2O + 4 electrons-
Electrophoresis Lab and Gel Electrophoresis of DNA: 12. Why is the DNA loaded in to the negative (cathode) end for gel electrophoresis?
DNA is negatively charged due to the phosphate (PO43-) backbone. When it is added to the cathode side, then they migrate from the negatively charged side of the gel box to the positive end, creating a potential energy difference which establishes an electric field through which the ions in the gel box fluid migrate. Since it is negatively charged, it will travel to the positive end since opposites attract.
DNA Extraction Lab: 5. What chemical inhibits DNAse by binding its cofactors?
EDTA (Ethylene Diamine Tetraacetic Acid)
DNA Extraction Lab: 4. What cation co-factor do DNAses need to break down DNA?
EDTA (Ethylene Diamine Tetraacetic Acid) binds divalent cations (Mg2+, Mn2+, Co2+). These are the cofactors to DNAse and other DNA cutting enzymes.
DNA Extraction Lab: 8. What is used to precipitate the DNA from the supernatant?
Ethanol is used to precipitate it. The DNA can be spooled because it excludes hydrophobic molecules. DNA cannot dissolve in alcohol.
Mitochondrial DNA Lab: 47. 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 DNA sequences. This should be especially true for siblings with the same mother as the mitochondria are exclusively inherited from the mother. Comparisons between ancestral humans and students revealed variable results depending upon the sequence of each individual student.
Restriction Enzyme Lab: 31. 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.
LARGEST-------- SMALLEST 5,500 1,700 900 800 600 500
PTC PCR Lab: 54. How could somebody have the 221 bp band and the 177 bp band as their genotype? Can they taste PTC? Explain how this "tasting" is different from the individuals in numbers 52 and 52.
Somebody can have the 221 band and the 177 band as their genotype if they're homozygous dominant or heterozygous. Homozygous dominant-they have 2 bands. One at 177 bp and the other at 44 bp. The 177 bp band will be just ahead of the uncut control, and the 44 bp band may be dim. The uncut band at 221 bp position should be fainter than the 177 bp band. Heterozygous- they have the 221 bp band that should be bolder than the 177 bp band. If 221 band is fainter, it wasn't digested completely.
PTC PCR Lab: 53. What is the significance of the 177 bp band in this lab? Can they taste PTC?
The 177 bp band indicates that you are homozygous dominant (TT) or heterozygous (Tt). You are able to taste the PTC.
PTC PCR Lab: 52. What is the significance of the 221 bp band in this lab? Can they taste PTC?
The 221 bp band indicates that you are homozygous recessive (tt) for the PTC gene. You are unable to taste PTC. You have a single band in the same place as the uncut control.
Transformation of E. coli Lab: 24. What is the role of the promoter found next to the GFP gene?
The ARA promoter activated in the presence of the sugar arabinose. When arabinose is present, the promoter will be turned on and cause the expression of the GFP gene and the production of the GFP protein in the cell.
Transformation of E. coli Lab: 18. What is the purpose of adding Calcium Chloride solution to the E. coli cells?
The CaCl solution shields the negative charge of phospholipids and allows the DNA to enter through the adhesion zone. It neutralizes the repulsive force within adhesion zones that would repel traveling plasmids. (stabilizes phospholipids)
the process of Polymerase Chain Reaction (PCR) that was a component of the mtDNA and PTC Labs: 34. 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?
The Chelex is an ion exchange resin that removes the polyvalent metal ions that are necessary cofactors for DNAses that also inhibit PCR reactions. The lysate/Chelex suspension was then centrifuged to eliminate cellular debris from the DNA containing supernatant. Cells lysed in presence of Chlex beads by incubating in boiling water.
the process of Polymerase Chain Reaction (PCR) that was a component of the mtDNA and PTC Labs: 39. Was the "pink stuff" identical for each of the three labs that we did? Explain.
The PCR primers had short sequences of nucleotides that were designed to bracket the region of DNA that was to be amplified. The pink stuff was different in the labs. The amplifications resulted in amplification of three different regions of our template DNA.
DNA Extraction Lab: 6. What chemical is used to dissolve the cell membrane?
The SDS (Sodium Dodecyl Sulfate or Sodium Lauryl Sulfate) dissolves the cell membrane and denatures proteins. It allows DNA to come out of the cells and into the solution
Transformation of E. coli Lab: 22. Why is there less bacteria growing on the LB+ amp plate than there is on the LB plate?
The ampicillin kills the plasmid DNA
2. 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?
The best place to deposit is at the bottom, then nothing will get stuck anywhere and all of the contents will be dispensed together and accurately measured in.
Transformation of E. coli Lab: 19. Why must the CaCl2/ E. coli mixture be incubated on ice?
The cold temperatures: -congeals the lipid membrane -stabilizes the phospholipids -makes the negative charge easier to shield
DNA Extraction Lab: 3. Why do we keep all materials and equipment cold during this lab?
The cold will slow the action of any DNAses or proteases (enzymes) which are sometimes released during the extraction and could ruin your DNA fibers.
Electrophoresis Lab and Gel Electrophoresis of DNA: 15. What do the color changes of phenol red indicate, specifically? At which end of the electrophoresis apparatus did each of these color changes occur?
The color changes of phenol red indicated changes in the pH levels. CATHODE: pink- acidic ANODE: yellow-basic
Mitochondrial DNA Lab: 44. Which portion of the mitochondrial DNA was amplified in this lab?
The control region was amplified. It accumulates mutations at approximately 10 times the rate of nuclear DNA. This results in unique patterns of SNPs. Control region is termed hypervariable since it does it fast.
the process of Polymerase Chain Reaction (PCR) that was a component of the mtDNA and PTC Labs: 40. What was the final substance we added to the PCR reaction mix? What is its purpose?
The final substance was the supernatant from DNA extraction. The purpose was to act as a template.
Electrophoresis Lab and Gel Electrophoresis of DNA: 13. How are the fragments of DNA separated at the end of electrophoresis? (Where are larger DNA fragments located relative to smaller DNA fragments)
The fragments of DNA are separated by turning on the electric current. The agarose gel then creates a microscopic mesh for the DNA to travel through. The larger pieces of DNA are move slower and not as far while the smaller fragments move faster and farther. The smaller the piece, the easier it is for it to travel through the matrix.
Restriction Enzyme Lab: 29. 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 how many times each restriction enzyme's recognition sequence appears in the sequence of the Lambda. # of fragments = +1 the number of cuts
Electrophoresis Lab and Gel Electrophoresis of DNA: 14. What was the purpose of phenol red in the "exploring electrophoresis" lab? Was this necessary when actually electrophoresing DNA?
The phenol red was used as an indicator for the pH changes occurring in the electrophoresis box. The phenol red is not a necessary component of electrophoresis; it was only used as an agent to visualize the changes that occur in the apparatus.
Mitochondrial DNA Lab: 45. How large is the piece of DNA?
The piece of DNA is 440 base pairs long
Transformation of E. coli Lab: 25. Explain how the presence of arabinose affects the expression of the GFP gene.
The presence of arabinose affects the expression of the GFP gene because then the promoter gets turned on and expresses the GFP gene and GFP protein production. When exposed to ultraviolet light, they glow green.
DNA Extraction Lab: 7. Once the cells are lysed, how are the proteins separated from the DNA?
The proteins are separated from the DNA through centrifugation. 2M NaCl denatures and precipitates proteins and therefore exposes the DNA.
Electrophoresis Lab and Gel Electrophoresis of DNA: 16. 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?
The reaction would be a shorter period of time because the reaction would grow increasingly unstable without the TBE buffer present.
the process of Polymerase Chain Reaction (PCR) that was a component of the mtDNA and PTC Labs: 33. Why was this saltwater solution spun in a centrifuge?
The saltwater solution was spun in a centrifuge so that it would harvest the squamous cheek cells to create a template DNA for the PCR reaction.
the process of Polymerase Chain Reaction (PCR) that was a component of the mtDNA and PTC Labs: 35. We then spun the boiled solution in a microcentrifuge. Was the substance that we wanted in the supernatant or the pellet?
The substance we wanted (the DNA) was in the supernatant.
the process of Polymerase Chain Reaction (PCR) that was a component of the mtDNA and PTC Labs: 41. If no template was added to the reaction, what kinds of products would be expected from the reaction?
There would be no products because the template is needed to produce the products. The PCR needs something to adhere to and follow for the code.
Transformation of E. coli Lab: 17. Why are rapidly growing bacterial cells needed for heat shock transformation?
They have regions in their cell membranes that are porous to DNA. These are called adhesion zones. They are large enough to have plasmids travel through them.
Restriction Enzyme Lab: 27. Restriction Enzymes are isolated from what type of organism?
They're isolated in bacterial cells
Mitochondrial DNA Lab: 48. If two brothers had 15 differences between their mtDNA sequences, what would this mean?
This would mean that they have different mothers.
Mitochondrial DNA Lab: 49. If two strangers had no differences between their mtDNA sequences, what would this mean?
This would mean that they have the same mother.
PTC PCR Lab: 51. Explain why we digested our PCR products with the restriction enzyme HaeIII.
We digested our PCR products with the HaeIII enzyme because it successfully cuts the 221 bp product into two pieces and the 44 bp and the 177 bp fragments.
the process of Polymerase Chain Reaction (PCR) that was a component of the mtDNA and PTC Labs: 32. The first step in the isolation of DNA for the PCR labs was to rinse our mouths out with a saltwater solution. What were we trying to extract from our mouths?
We were trying to get template DNA found in our squamous cheek cells (mitochondrial DNA)
Mitochondrial DNA Lab: 46. Who did you inherit your mitochondrial DNA from?
You inherit your mitochondrial DNA from your mother only.
Transformation of E. coli Lab: 26. If a green glowing bacterial colony is grown on a plate lacking arabinose, what color would you expect it to appear?
You wouldn't expect it to be any color because without arabinose, the same cells that contain the GFP gene but don't express the GFP protein wouldn't glow green in the ultraviolet light.
the process of Polymerase Chain Reaction (PCR) that was a component of the mtDNA and PTC Labs: 38. What was the "pink stuff" that was added to the PCR bead?
mtDNA primer mixed with loading dye (primer mix) PRIMERS
1. What would be the correct setting on the pipette to measure the following volumes: (microliters) a. Blue- 100-1000 ul pipette: 500 ul, 250 ul, 760 ul b. Yellow- 2-20 ul pipette: 2 ul, 25.5 ul, 5 ul
~Blue line goes 1000s, 100s, 1s a. 500- 050 250-025 760-076 ~Yellow line goes 10s, 1s, 0.1s b. 2-020 25.5-255 5-050
