3 Genes Flashcards

Question

What can you get genomic dna from

Answer 1

Cell culture Animal tissue Blood Bacteria Plants

Answer 2

Same steps except you precipitate out the dna and collect that whereas plasmids stay in solution and are collected In solution

Answer 3

Sample collection and preservation Lysis: - chemical - mechanical - enzymatic Purification: - large particles /molecules/cell debris separated by centrifugation (DNA/RNA stay in the supernatant) - many method of them purifying the dna : phenol-chloroform extraction followed by ethanol precipitation

Answer 4

Using RNases Uses trizol to separate the two

Answer 5

The collection process varies based on the cell/tissue type Can collect from culture, from a cave finding ancient human dna in dirt

Answer 6

Cell cultures: need to centrifuge to remove the growth media Animal or plant tissue: need to cut up into smaller samples Blood: needs anticoagulant when collecting Environmental samples: filtered to concentrate organic matter or remove large inorganic particles

Answer 7

Types of lysis depends on the sample Manual grinding: - for tough tissues (plants, insects) or large samples Mechanical blending: - complex tissues like liver and muscle Freeze thaw: freezes sample and make crystals that shear cells Bead mills/bead beatinf: - rapid agitation of sample with beads - for tough smaller tissues, bacteria, yeast, fungi, complex matrices (like soil and feces) Sonication: - high frequency sound waves to shear cells - for bacteria and yeast

Answer 8

Detergents: - SDS (breaks cell membranes) - disrupts interactions between lipid molecules Hypotonic solution: - water goes into the cell via osmosis until it bursts Chaotropic agents: - urea and guandine - disrupts the hydrophilic nature of water which lets the hydrophobic cell walls become soluble EDTA: - chelates Mg2+ - disrupted bacteria cell walls - chelating Mg blocks DNase activity (which keeps dna intact since DNase degrade it)

Answer 9

Lysozyme, mutanolysin, Lysostaphin: - cleave peptidoglycan linkages (peptide bonds) that are in cells walls of gram postive bacteria (so break down cell walls) Proteinase K: stops proteases - a broad spectrum protease that still works in SDS, EDTA, or DTT - digests nucleases

Answer 10

EDTA stops their function Proteinase K digests them

Answer 11

They’re both in the supernatant but want to remove RNA using RNases because if pricier with the dna is messses up quantiation of dna because it also absorbs light at 260 nm

Answer 12

After cell lysis, centrifugation the sample to pellet large cell debris The supernatant is collected for next steps

Answer 13

Mix the cell lysate with phenol and chloroform, centrifuge, Order of stuff: supernatant (aqueous layer) has DNA and RNA - interface has coagulated protiens - bottom (organic layer) has phenol The phenol denatures protiens and causes precipitation of the protien impurites Chloroform increase the efficiency of phenol for denaturing protien and it denatured lipids. It also allows proper separation of organic and aqueous phases

Answer 14

This is to precipitate out the DNA in the supernatant, used as a final clean up or to concentrate dna Ethanol and sodium acetate ppt the nucleotides Genomic dna in the pellet

Answer 15

Silica filter extraction Magnetic bead extraction IEC CTAB extraction

Answer 16

Cell lysate on silica beads, the DNA (or RNA) bind the silica membrane in HGIH SALT elute the DNA with low salt (or water) Ethanol washes do not elute the DNA

Answer 17

Add the beads to the cell lysate DNA in the lysate binds the silica particles or silica coated magnetic beads Same principle as silica column (resuspend silica in water then centrifuge to elute the dna)

Answer 18

Put lysate in IEC with low salt As salt increases elution happens I. This order: 1. Protiens 2. RNA 3. DNA

Answer 19

Cetyltrimethylammoniumbromide It’s a detergent that denatured polysaccharides (sugars) but ppt the DNA at same time good for plant tissue because of their high carbohydrate content DNA in pellet

Answer 20

Similar to genomic dna extraction but with exceptions For gDNA, collecting all dna, but for RNA are you Collleting total or polyA rna? - if working with eukaryotes need to know if extracting poly a rna , if yes you use oligo (dT) to isolate it (bind the A) RNA is easily degradable: - due to 2’ OH - so you have to work faster and at lower temp RNases are very ubiquitous: they’re everywhere so you need to be sterile when doing extraction - use RNase inhibitors (DEPC) - use RNase free reagents (DEPC-treated), supplies, and equipments (pipettes) - use RNase away (from thermofusher)

Answer 21

You use the trizol reagent (which includes phenol) as an alternative to phenol chloroform extraction With phenol chloroform extraction, bith RNA and DNA in aqueous phase, But with trizol, the dna goes into the organic phase , RNA into the aqueous phase Three layers: - aqueous (RNA) - interphase and organic (DNA and protiens) But can use slikica filters, magnetic beads, IEC, and CTAB all for RNA extraction as well

Answer 22

Agarose gel electrophoresis Quantitation; - spectrophotometry - fluorimetery - bioanalyzer

Answer 23

Can use to determine general quality but not quantity of gDNA Good samples: - minimal smearing (because smearing is dna of a bunch of diff sizes) - all bands same size (means no dna degradation) - no faint bands at bottom of gel (usually that means there’s RNA contaminants) - no chunks in wells (means no remains protien/impurites

Answer 24

Critically important for library perpartation in genome seqeluencing

Answer 25

Good enough for wuiatify you gene if the gene is just being used for PCr , if want really accurate it’s not good enough (like if want to do genome sequencing Use bioanalyzer or fluorimeter if want dna for genome sequencing

Answer 26

Can see dna size and quantity at same time Size is x axis, y is intensity kf peaks (quantity) Shows peaks Has DIN (dna integrity number) which is a measure of how degraded a genomic dna sample is

Answer 27

Gel electrophoresis: - works for RNA but the RNA secondary structures interfere with migration because not linear (this is why we use denaturing agent) - know that RNA preps are good Kd see 28s and 18s bands in gel Spectrophotometry: - works normally Flurorimetry: - need RNA specific fluorophore - only find quantity [] and not quality Bioanalyzer: - rna specific chips needed

3 Genes Flashcards

(52 cards)