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BIOL Exam I - Ex. 5
Restriction Digests, PCR, and Bacterial Transformations
| Question | Answer |
|---|---|
| What's a plasmid? Which plasmid did we focus on in exercise 5? | - small ring of DNA within bacteria - distinct from, and replicates independently of, the bacterial chromosome - pGLO |
| What are five important components of pGLO and what do they provide to the bacteria? | - ori: origin for DNA replication - bla: codes for enzyme that provides ampicillin resistance - GFP gene: codes for green fluorescent protein which provides fluorescence under UV - multiple cloning site (MCS): where restriction sites are |
| The pGLO plasmid is ___ base pairs long, however, in its supercoiled form, it runs on an agarose gel in the ___ range. | - 5,371 - 4,200-4,500 |
| One of the components of pGLO is the araC gene. What does it do? Explain how arabinose is involved. | - codes for the araC protein - in the absence of arabinose (a sugar), the protein prevents the transcription of the GFP gene - if arabinose is present, the protein cannot prevent transcription of the GFP gene |
| Why did some of the E. coli we observed glow? | - E. coli were transformed (took up pGLO) - these bacteria were placed on a media containing arabinose - the presence of arabinose allowed for transcription of the GFP gene (araC protein overridden) - GFP produced - bacteria fluoresces |
| What does it mean for bacteria to be "chemically competent?" How is this done? | - bacteria have been made to take up plasmids from the environment - done by treating the bacteria with a high concentration of calcium ions (makes their phospholipid bilayers "leaky") |
| In exercise 5 you were given both chemically competent E. coli and pGLO plasmid with the GFP gene already inserted. What did you do with those? | - transformed the chemically competent E. coli WITH the pGLO plasmid - low success rate |
| How do you know which bacterial cells have been transformed with the pGLO plasmid? | - only those bacteria that have been transformed will survive on plates with ampicillin in the media - (selection of transformed bacteria) |
| On which plate(s) would you expect transformed bacteria to grow: 1(+LB,–AMP,–ARA), 2(+LB,+AMP,–ARA), 3(+LB,+AMP,+ARA)? Explain. | - on all three - untransformed bacteria will be dominant on plate 1, resulting in a white "lawn" - only transformed bacteria will be on plates 2 and 3 |
| On which plate(s) would you expect GFP to be produced: 1(+LB,–AMP,–ARA), 2(+LB,+AMP,–ARA), 3(+LB,+AMP,+ARA)? Explain. | - only on plate 3 - it contains arabinose, so araC will be overridden, resulting in the transcription of the GFP gene |
| On which plate(s) would you expect bacteria to fluoresce: 1(+LB,–AMP,–ARA), 2(+LB,+AMP,–ARA), 3(+LB,+AMP,+ARA)? Explain. | - only on plate 3 IF ultraviolet light is shined on it - fluorescence only visible under UV |
| Will transformed bacteria always fluoresce? | no, only when arabinose is present AND ultraviolet light is shining on it |
| genotype + environment = ___ | phenotype |
| restriction sites | - locations on a DNA molecule containing specific sequences of nucleotides which are recognized by restriction enzymes - where foreign DNA can be inserted |
| restriction enzymes | - naturally occurring enzymes produced by bacteria that cut DNA molecules - each restriction enzyme only recognizes a very specific DNA sequence, where it binds and then cute the double-stranded DNA |
| If BstEII-HF and ECO RI-HF act on pGLO, what is the result? | two fragments of DNA (because two cuts) |
| What is PCR? How does it make copies of targeted regions of DNA? | - the polymerase chain reaction - thermal cycling (cycles of repeated heating and cooling) |
| primers | - short DNA fragments that contain sequences which flank the targeted region of DNA - the starting points that DNA polymerase use |
| DNA polymerase | - enzymes that create DNA molecules by assembling nucleotides, the building blocks of DNA - essential to DNA replication and usually work in pairs to create two identical DNA strands from a single original DNA molecule |
| What are the three steps of PCR? | - denaturation - primer annealing - extension - (steps repeated to yield the necessary number of copies) |
| denaturation | - mixture heated to 95°C - hydrogen bonds break - two strands of DNA separate |
| primer annealing | mixture quickly cooled to 55°C to allow short, single-stranded primers to bond to their complementary sequences of target DNA |
| extension | - mixture heated to 72°C - Taq polymerase synthesizes the complementary DNA strand from the dNTPs, starting at the primers |
| What region on chromosome 1 did we examine using PCR? | the D1S80 locus |
| There is a ___ base pair repeat unit on the D1S80 locus. The allele corresponds to the number of times the unit repeats. There are ___ different alleles in the human population. | - 16 - 28 (14 to 41 repeats, 369 to 901 base pairs) |
| You have two alleles for the D1S80 locus. You can either be homozygous or heterozygous. Most people are ___. You will determine your genotype using ___ and ___. | - heterozygous (>80%) - PCR and gel electrophoresis |
| To isolate and determine the length of a target region why are two primers needed? | so that a DNA polymerase can attach to the 5' strand and one can attach to the 3' strand |
| How do you purify genomic DNA? | BIND (DNA to matrix) -> spin -> WASH (solution to remove impurities)-> spin -> ELUTE (DNA from matrix) -> spin |
| In exercise 5, what was in the master mix? | - Taq polymerase - MgCl2 - dNTPs - buffer |
| gel electrophoresis | a method for separation and analysis of macromolecules (DNA, RNA and proteins) and their fragments, based on their size and charge |
| Plasmid genes are not essential to bacterial life, but often provide a ___ to the cells that possess them. | selective advantage |
| colony | a small circular lump of billions upon billions of cells |
| EcoRI | - restriction enzyme used extensively in genetic engineering - the first restriction enzyme isolated from E. coli |
| During gel electrophoresis, nucleic acids move towards the anode. Why? | the fragments move toward the positive electrodes (anode) because of the negative charge carried by their sugar-phosphate backbone |
| During gel electrophoresis, large fragments of DNA move more ___ through gel while the smaller fragments move more ___. This is how distinct bands form on the gel. | - slowly - quickly |
| The speed of a circular plasmid will depend on whether the plasmid is supercoiled, nicked/relaxed, or linear. Order these from what will move the slowest to the fastest in gel. | - nicked/relaxed circle (because it's floppy) - linear (because it got broken) - supercoiled (because it's compact) |
| DNA ladder | - a solution of DNA molecules of different, KNOWN length; a reference - used to determine the actual length of molecules (number of base pairs) |
| Taq polymerase | - a DNA polymerase isolated from extreme thermophiles; heat resistant - used by scientists to artificially replicate DNA in vitro (PCR) |
| What does a typical PCR vial contain (5)? | - template DNA - Taq polymerase - dNTPs (monomers of DNA) - primers (starting point for the enzyme) - buffer solution + cofactor Mg2+ (provides a suitable chemical environment for the enzyme) |
| primer dimer | - a potential by-product in PCR - consists of primer molecules that have attached (hybridized) to each other because of strings of complementary bases in the primers - results in amplification of untargeted DNA |
| The D1S80 locus is a short tandem repeat (STR) region on human chromosome 1. What are STRs? | short (100-300 bp) regions of the human genome that have undergone duplication or deletion over the course of evolutionary time resulting in... - ...unit repeats of different lengths (called variable number tandem repeats [VNTRs]) |
| If you are ___, the fragments of different sizes will separate, forming distinct bands on the gel. If you are ___, you will only see one band on the gel. | - heterozygous - homozygous |
| What protocols did you complete during exercise 5 (5)? | - digest a plasmid with restriction enzymes - transform bacteria with a plasmid - streak bacteria on agar plates - swab my cheeks - isolate my DNA |
| engineered plasmid | - a vector that can be used to insert a useful gene into a bacterial cell (like resistance to an antibiotic) - a circular DNA molecule found in bacteria that replicates independently of bacterial chromosomal DNA; has restriction sites |
| What does it mean for a cell to be competent? | the charges on the phospholipid bilayer are neutralized by CaCl2 to allow the cell to uptake DNA |
| What does it mean that a bacterial cell has been transformed? | a plasmid has been inserted into the cell |
| How should agar plates be stored to allow effective bacterial growth overnight? | - parafilmed - rubber-banded together - properly labled on the bottom periphery - placed inverted in the 37°C incubator |
| The main purpose of repeating the three steps (denature, anneal, extend) in a PCR protocol for multiple cycles (typically 30-40) is to ___. | amplify the amount of the targeted DNA sequence |
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jessica.gvc
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