post lab discussion: exp 7

Question 1

what are the different types of procedure of DNA isolation

Answer 1

1. Cell disruption or cell lysis.
   a. Physical cell disruption
   - physical force (smash it)
   b. Chemical cell disruption
   - chemical reagent, in order to remove the DNA [dissolve the cell walls and membranes to release the DNA]
2. Removal of membrane lipids.
   a. Detergents or
   surfactants [work like dish soap, breaking apart the greasy membrane to free the DNA]
3. Denaturation and removal of proteins.
   a. Protease
   [Proteins are everywhere in the cell, so we need to get rid of them to isolate pure DNA.
   Protease: This is an enzyme that breaks down proteins, leaving the DNA behind.]
4. Removal of RNA.
   a. RNAse
   [Cells also have RNA (another genetic material), so we use:
   RNAse: This enzyme eats up the RNA, leaving only the DNA]
5. Purification.
   - make sure that it is DNA lang
   a. Ethanol precipitation [Adding alcohol to make DNA clump together and sink to the bottom]
   b. Phenol -chloroform extraction [ to separate DNA from everything else]
   c. Mini -column purification [using a filter to trap the DNA and wash away impurities]

Question 2

what are the contents of homogenizing solution

Answer 2

ethanol
NaCl
protease
SDS
tris-buffer

Question 3

this facilitates Na+ interaction with DNA’s phosphate groups.

Answer 3

ethanol

*as the phosphate group of dna is negatively charged, the positive charge of the Na neutralizes the charge of dna

Question 4

Na+ binds with DNA forming a precipitate (salting out method).

Answer 4

NaCl

*salting out method
making the precipitate come out, through the sodium ions neutralizing the dna phosphate grp

Question 5

breaks down proteins bound to DNA (better DNA yield)

Answer 5

protease

*breaks donwn of proteins bound to dna, so that the isolation is dna

Question 6

breaks down the phospholipid bilayer of the cell particularly that of the nucleus (nuclear membrane) and the proteins in it, allowing for the release of DNA.

Answer 6

SDS

*a detergent

Question 7

resuspension buffer making the DNA stable and viable

Answer 7

tris-buffer

Question 8

what is the purity of DNA

Answer 8

A260nm/A280nm = 1.8 – 2.0

Question 9

what are the methods of detection of DNA and RNA concentration

Answer 9

a. Linear regression
- through calcu or excel

b. Optical Density

c. Densitometry

Question 10

what are the purpose of DNA extraction

Answer 10

a. Identification
b. Genetic disorders
c. Environmental studies

Question 11

what are the other specimens for DNA extraction

Answer 11

human peripheral
blood
buccal cell
hair follicle
urine and organs
plants, animals and bacterial samples.

Question 12

these are indeed candidate sources of genes of medical or industrial importance.

Answer 12

dna extracts

Question 13

what other techniques that could be employed to harness this potential

Answer 13

PCR - Polymerase chain reaction
Purification steps
Sequencing
- bioinformatics (more on genome)
Recombinant dna technology
- restriction digestion
- ligation

Question 14

what are some of the ethical considerations regarding extraction of DNA from human source

Answer 14

• Participants properly and clearly informed about the
  study.
• Background of the study
• Protocol
• Possible adverse effects.
  
  • Method of extraction.
  • Exposure to reagents.
• Informed consent.
• Knowledge and permission that a biological sample
  is to be taken.
• If sample will be stored at a given period of time or not.
• If further experimentations to be done using the sample in the future.
• Participant is informed about the outcome.
• Interventions

Question 15

what are the modifications needed depending on the nature of sample sources

Answer 15

• Contaminants (i.e. proteins and carbohydrates)
• Cellular components (i.e bacteria and plants cell wall) - depending on how u store the dna samples
• Purification of DNA extracts for downstream applications.

Question 16

what are the possible source of dna and considerations

Answer 16

• Fossil
• Microbial
• Ecto and Endo parasites
• Histopathological
• Crime Scene & Forensics
• Botanical

Question 17

Used for determining the presence of nucleic acids.

Answer 17

Dische’s Test

Question 18

what will the dische’s test detect

Answer 18

Detect the deoxyribose of DNA and will not interact with the ribose in RNA. (Green color for RNA)

*The intensity of the blue color is proportional to the concentration of DNA. [the greater the conc in the sample, the bluer]

Question 19

Acidic conditions convert deoxyribose to a?

Answer 19

to a molecule that binds with diphenylamine to form a blue complex.

Question 20

what is used to assess DNA purity

Answer 20

UV-Vis Spectrophotometer

Question 21

what type of UV VUS did we use

Answer 21

double beam UV-VIS (2 light source)
- has 2 slots for cuvettes (quartz cuvettes)

Question 22

why is quartz cuvettes used and not plastic cuvettes

Answer 22

over time, the plastic cuvettes gets old and it will become yellowish
hence, might confuse the UV-Vis as they are very sensitive to color

Question 23

how do you get the purity of the dna

Answer 23

divide it from one another

absorbance u get from 260 and absorbance from 280 (there is specific range to say if your dna is pure. its btwn 1.8 to 2.0)

• lower than 1.8 (presence of proteins, as the proteins absorb strongly near 280)

Question 25

what is the generally accepted as “pure” dna

Answer 25

approx. 1.8-2.0

• if below 1.8
  may indicate presence of proteins, phenol or other contaminants that absorb strongly at or near 280nm
• if more thn 2.0
  may indicate presence of RNA
• 0.6
  for pure protein

Question 26

what are the following 5 nucleotides that represent the 260/280 ratios estimated for each nucleotide if measured independently

Answer 26

guanine 1.15
adenine 4.50
cytosine 1.51
uracil 4.00
thymine 1.47