Bisc 357 Updated Actual Questions And Correct Answers

BISC 357 UPDATED ACTUAL QUESTIONS AND
CORRECT ANSWERS

Question:
1. isolated DNA needs to be (3 things)

Answer:
free from contaminants
be in frag sizes larger than will be cloned
concentrated enough to be manipulated in ul volumes

Question:
2. steps of DNA purification

Answer:
1. mechanical disruption of tissue to break cells and free DNA
2. chemical disruption of cells to access DNA using extraction buffer
3. : Stepwise removal of components other than DNA:

Question:
3. whats in extraction buffer

Answer:
- Tris buffer, pH 8, supports DNA stability
-EDTA, chelates (binds) divalent cations, including Mg 2+, which is a cofactor for
DNA-degrading DNA nucleases (DNases) inhibits DNA degradation
-Strong detergent, to disrupt cell membranes and secondary structures of
proteins, including DNases releases DNA and inhibits DNA degradation (typically
sodium dodecyl sulphate; SDS)
NaCl, helps solubilize DNA, also required for subsequent DNA precipitation

Question:
4. Stepwise removal of components other than DNA (5)

Answer:
1. Pellet large particles by centrifugation. Move clear solution with dissolved DNA
(and other materials) to new tube.
2. Remove proteins by extraction with nonpolar organic solvent and/or by
conditions that ppt proteins but not DNA
3. Remove RNA by RNAse enzyme degradation
4. Remove smaller metabolites by salt and alcohol-induced DNA pptation. After
centrifugation, solution with metabolites is removed and pelleted DNA is
resuspended in smaller volume of buffer.
5. Concentration or dilution of DNA into working concentration. Step 4 also
concentrates DNA

,Question:
5. how to remove proteins from DNA solution to isolate
DNA

Answer:
by extraction with nonpolar organic solvent and/or by conditions that ppt
proteins but not DNA

Question:
6. nonpolar organic solvents used to extract proteins from
DNA solution

Answer:
chloroform and phenol

Question:
7. conditions that ppt proteins but not DNA

Answer:
high salt
low pH
low temp

Question:
8. proteinase K

Answer:
degrades proteins

Question:
9. adsorption chromatography method steps (3)

Answer:
1. Prepare crude lysate
2. Adsorb to silica surface
3. Wash away residual contamination using solution that favors DNA binding to
silica matrix
4. elute DNA

Question:
10. chaotropic salts

Answer:
salt that disrupts H bonds
Also disrupts hydration shell around DNA, allowing cation bridge-binding
between silica and negatively charged DNA

, Question:
11. How does extraction Buffer composition favors DNA and
RNA adsorption to silica

Answer:
- Low pH
- High ionic strength
- Chaotropic salt (guanidinium)

Question:
12. how is Elution buffer unfavorable to surface binding

Answer:
high pH
low ionic strength

Question:
13. how RNAse remove unwanted RNA

Answer:
catalyze the hydrolysis of RNA into smaller components (NUs or smaller stretches
of RNA)

Question:
14. low and high DNA A260/A280 ratio meaning

Answer:
high: sample is either contaminated by protein or a reagent such as phenol or that
there was an issue with the measurement
low: phenol contamination

Question:
15. low and high DNA A260/A230 meaning

Answer:
Higher: contamination of phenol, trizol, ghcl, and guanidine thiofidvnssa dkj
lower: carb carryover

Question:
16. how to check for degradation of DNA

Answer:
run sample through an agarose gel (0.7- 1.5%) to examine the length of DNA
fragments and extent of DNA degradation.