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BIOL Exam I - Ex. 5

Restriction Digests, PCR, and Bacterial Transformations

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
Created by: jessica.gvc