determining DNa concentration and purity lab

should also study -solution problems -best instrument -2 replicates

key molecules in study of life

• DNA

• RNA

• carbohydrates

• lipids

• proteins

very small so it is imposible to see

what can we use to see the small molecules of life

• electron microsope

• NMR (nuclear resonance spectroscopy)

• MS (mass spectroscopy)

• Ir (infared spectroscopy)

• GC (gas chromotorgraphy)

• HPLC (high performance liquid chromotography)

• UV-vis (Ultraviolet visible spectrophotmetry)

objectives of the lab

• perform DNA quantification and purity assessment

• understand underlying concepts of spectrophotometry

• create and interpret standard curve

• determine DNA concentration of unknown samples

basic steps

• Add 500microliters of digestion extraction buffer (detergent)

  • break down cell membrane

• 50 microliters of 20mg/ml proteinase K

  • breaks down proteins

• swab mouth for thirty seconds and swirl in the tube

• hot water bath for 20 mins at 65 degrees

  • activate enzymes

• Add potassium acetate

  • cause DNA to preciptate and stay tightly bound

• put on ice for 15 minutes

  • DNA precipates in low temps

• centrifuge for 7 minutes

  • brings all the waste to the bottom

• pour off liquid into another microtube and discard pellet

• Add 300 micro liters of 95% ethanol and mix

  • removes remaining salts and allows DNA to return to solution in TE buffer

• ice until next class

  • avoid decay since DNA is harmed at certain temps

• centrifuge for 10 minutes and pour off all liquid minus the pellet

• at 1000 microliters of 70% isopropanol and centrifuge for 10 mins

  • iso- washes potassium acetate salts and removes any remaining proteins from solution

• rehydrate with TE buffer

• spectrometer for 234, 260, 280, 320nm

• clean

why is it important to determine the quantity and purity of DNA in a lab setting

can be used for gel electrophorisis, and PCR tests

GE- spreads out

PCR- replicates

260

optimal absorbance for DNA and nucleic acids, on hte UV-vis, measures DNA concentration

280

optimal absorbance for proteins

pure RNA, DNA, and protein ratios

• 2.0

• 1.8

• 0.6

  respectivly

if we had treated with RNAse

260 would decrease and ratio of 260/280 would go down. Less absorbance

did not use proteinase K

280 number would increase causing a smaller ratio.

94 degreese on that mf

260 would go down, high temperatures degrade DNA samples so the ratio would decrease and less would be absorbed.

if we used plant cells what else should we do?

smash cell membrane with hands or use cellulose

UV-VIS spectrophometer

First was WW2 to determine vitamin content of food supply given to the troops

Used to measure many molecules

bombarding molecule with specific wavelenghts of life

determines how much is absorbed by seeing how much hits the detector, More absorbed the more concentrated.

Use 260/280 to find purity of a DNA sample

Put sample in cuvettes

• made of plastic of quartz

• blank- tube of TE with absorbance of 0

parts of a uv-vis

light source

entrance slit

dispersion device

exit slit

sample

detector

absorbance spectrum

when multiple wavelenghts are examined

beer-lambert law

Absorbance=molar absorptivity L/mol cm * path length cm * concentration in Mol/L

why is purity important

• asses contaimination

• afect PCR and Gell electrophorisis

• affects digestion involving restriction enzymes, proteases degreade DNA

what does tris do in TE buffer

maintains PH

what does edta do in te buffer

solubilize nucleic acids while protecting them from enzymatic lysis