biol 3110L rev for test on 11:10

Biol 3110L rev for - BIOL 3110L REVIEW FOR TEST 3-know dif buffers for each step and different salt buffers(for PGLO vs GFP plasmid purification

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BIOL 3110L –REVIEW FOR TEST 3 -know dif buffers for each step and different salt buffers? (for PGLO vs GFP) plasmid purification buffers versus protein buffers protein buffers: QT buffers for washing through ciatin tips? GFP buffer: (see GFP purification document) -equil. buffer: medium salt buffer; before put sample in put buffer so it gets sample ready?. .. (QBT) -wash buffer ---medium buffer; wash out stuff that’s loosely bound to gel (QC) -QF (elution buffer) –low salt; decrease exposure to hydrophobic regions… -binding buffer—high salt buffer; use them b/c they help expose hydrophobic regions of protein and give protein more ability to bind to gel? *protein buffers not mentioned in protocols?. ... -know all the steps for PCR and the different temperatures for PCR Plasmid buffers: -P1 (resuspension buffer), P2 (lysis buffer/detergent buffer), P3 (neutralization buffer) QC, P1: has a chemical called EDTA and pulls metal ions out of solution and without calcium… pulls all of calcium out so things can’t stick to e/o P2: contains some kind of detergent with hydrophobic and hydrophilic portions. So It make miscelles with bacteria cell membrane so it ends up lysing them. P3: a neutralization buffer; contains potassium acetate, does not adhere to ?? prevents proteins from sticking to dna; so can isolate JUST the dna P2 lets you get dna out of cells… Calculation: M1V1=M2V2 final volume of buffer: always 400 mL ethidium bromide = 5 microliters
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ex: 50 (v) = 1(400mL) v= 8 mL *if he doesn’t say anything then assume it’s a 1% gel. **Lab report: digested two plasmids and recombined them…. Transformation: methods used—heat shock and calcium chloride --50% of protocols say DON’T heat shock BAM III
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1.) PCR-based VNTR Human DNA Typing: general facts: polymorphic DNA = chromosomal regions that vary widely from individual to individual RFLP (Restriction Fragment Length Polymorphism) = results when DNA is digested with a specific restriction enzyme and a probe is allowed to hybridize to a specific region; examination can be done with PCR or southern blot Most common sources for DNA typing: blood, hair and saliva Cells collected must be treated to release their DNA into solution. In forensic laboratories, specimens collected from crime scenes are treated by various methods to lyse the cell membranes and release the DNA. o Then the cells are resuspended in a chelating agent ( removes cellular cations that inhibit PCR ). o DNA then undergoes RFLP or AMPFLP analysis. o Profile obtained is then compared with analysis of DNA from the victim or suspect. PCR= polymerase chain reaction o invented in 1984 by Kary Mullis; awarded a Nobel Prize in 1994 o Why is PCR used so much? b/c its ease of use and its ability to amplify DNA o PCR amplification uses the enzyme Taq polymerase (stable at high temps) o Also used in PCR mixture: 2 primers and target DNA (extracted DNA template) **Steps to PCR reaction aka “one PCR cycle”: 1.) Target complimentary DNA strands are melted/separated from each other at
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This note was uploaded on 01/19/2012 for the course MUSC 2960 taught by Professor Arvinscott during the Fall '11 term at University of Georgia Athens.

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Biol 3110L rev for - BIOL 3110L REVIEW FOR TEST 3-know dif buffers for each step and different salt buffers(for PGLO vs GFP plasmid purification

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