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18 Cards in this Set
- Front
- Back
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Microbial Species aren't so easily defined because... |
1. It's entirely possible for 2 unrelated microorganisms to have similar phenotypes and biochemical traits. 2. Biochemical traits can be easily lost by mutation without changing the identity of the species... E.Coli with lactose mutation is still E.coli 3. Labor intensive and specialized tests would need to be performed to identify, groups of bacteria and different tests need to be used for different organisms. Classification by phenotypic properties often made understanding evolutionary relationships among bacteria difficult.. just because 2 organisms have similar phenotypes doesn't mean they are evolutionarily related |
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Why did Carl Woese test the ribosome? |
1. Universal - being present in all life forms, ribosome is most ancient function in biology 2. Conserved - cannot change too quickly or change too slowly. rRNA is ideal because it contains regions of great conservation that almost never changes, and other regions of hypervariability.. Thus it is possible to differentiate using hypervariable regions. 3. Not transferrable - bacteria can share DNA, and not just amongst own species. |
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What is the gene associated with Woese's studies? |
16s rRNA |
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What is PCR? Function |
Polymerase Chain Reaction. Used to amplify a region of DNA. It does not purify. that should be done beforehand. |
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What do we use to break open bacterial cells and release DNA along with other cell contents? |
NaOH and heat! |
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What do we use with the PCR products? |
Gel electrophoresis and sequencing |
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What can you use to purify DNA from a mashed up strawberry AT HOME? In the lab? |
Home = Dish Soap, Rubbing Alcohol, Salt Lab = NaOH, EDTA, EtOH |
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Why could sequencing be messed up? |
-Contamination by more than one template -Too much DNA template (arguable might be the PCR product wasn't expressed and amplification came from template DNA) -More than one primer (recognizes and amplifies more than one size) -low annealing temperature (causes nonspecific priming of primer to the template strand) |
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Your Sequence says 100% and 99% matches all over the place.. what is your isolate? |
Inconclusive. It's a good route to go and to assume but you can never say for 100% certain that's what it was. |
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If you have two runs that have the same base pairs, what can you say about those two isolate species |
you cannot tell.. just because they have similar PCR product does not mean they are the exact same. |
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what happens if there is more than one band in each lane? |
Low annealing temperature... contamination.. non specific amplification |
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Where are the primer dimers on the agarose gel? |
Towards the end on the positive side.. farther is the degraded RNA |
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What's the difference between enrichment and purification? |
Enrichment is for a type of microbes Purification is for one species |
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How many nucleotides are typically in the 16s rRNA template |
1500bp |
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What is the first step to DNA amplification? |
DNA extraction via lysis... we use naoh and heat |
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What is the problem with polymerase? |
it is temperature dependent. so to fix it we use TAQ.. which is temperature permissive |
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What is the process for amplification? |
1. Denaturation: it's heated briefly to separate the DNA into two strands 2. Annealing: cooled to allow primers to form hydrogen bonds with the ends of the target sequence. 3. Extension: DNA polymerase adds dNTPs to 3' end of the primers. This whole process yields 2 molecules. Another round yields 4, another round yields 8... after the 3rd round two of the 8 molecules match the target sequence |
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How to check for successful amplification |
Do you have the correct fragment size on the agarose? 1500 bases? Do you have enough DNA? nanodrop of qbit |
