Materials that bind ● Silica

DNA: ○ most common

DNA/RNA EXTRACTION AND VISUALIZATION
○ found in columns (column method)
○ can bind to DNA, does not wash out it precipitate in the solution
Recall: ● chelex
● DNA is made up of sugar phosphate backbone
● Hydrogen bond connect the nucleotides DNA-binding conditions:
● Cytosine - Guanine
● Adenine - Thymine
Occurs in Acidic ● At this low pG there are lots of free protons (H+) to neutralize silica in the
● Genome: entire genetic information
pH membrane
● Gene: short part of DNA that codes for a particular trait
● Can only makes proteins
● Nucleus contains DNA (eukaryotes) With high salt ● Guanidinium (salt)
● this chaotrope loosens up the water coat surrounding DNA,
● denatures the DNA Pol & helps shield the DNA’s negative charge so that
If u want to study the DNA/RNA the first thing you will do is get it (extract) from the source
the DNA & silica can get close & form more extensive bonding
Samples
● Should contain cells with nucleus where the DNA or RNA is found (cheek cells for paternal test) Presence of ● Helps them find each other
● You don't need to match sample to sample (example if you want to match a semen sample use the Isopropanol ● kick off the water
cheek cells because we are after the DNA not the sample)
d) Wash ● removes other impurities to purify the DNA
● Done 3 times to make sure all impurities are removed
● esp. RNA

Wash solutions: ● Buffer with ethanol

● Wash buffer (1 M Tris, 5 M NaCl, 0.5M EDTA, Abs. Ethanol)

Of course there are DNA extractions that do not involve washing, usually
there is only limited downstream applications. DNA cannot be stored so we
use DNA right after the extraction.

A. DNA Extraction Crude

● Boiling method
1. Sample Preparation ● not removing impurities usually in the lab, it will be used immediately
after preparation of DNA for pcr or freeze it immediately so that the DNA
will not be degraded
a) Cell Lysis ● break down of cell membranes and nuclear membrane
● Detection for specific gene
● most important part of DNA extraction
● failure in DNA extraction always relates on failure of cell lysis
● fungi is the most difficult organism to lys because of its thick cell wall Wash Conditions: Ethanol
● Dissolves salts, but not DNA, so you can wash the salt away
● DNA is not being dissolved because it is attached to the silica
Lysis solutions: ● NaOH ● But also can interfere later so make sure you spin twice to remove excess!
● SDS (sodium dodecyl sulfate)
● 6M Guanidine HCl
○ Effective in extraction in different microorganisms e) Elution ● dna is now removed from the particle where it is bound in order to get into
the solution
● release of DNA from the material to which it is bound
b) Protein removal ● to purify the DNA so that there is no other proteins or enzyme that can
(optional) damage the DNA
● uses Proteinase K Eluents: ● TE(Tris-EDTA)buffer
○ Reagent use for removing of protein debris after cell lysis ● Distilled water
○ Some kits do not use Proteinase K (proteins are just washed)
eluate ● solution that comes out of elution
c) DNA binding ● DNA binds to specific materials
● Binds to the particles that cannot be washed out Elution Low salt, higher pH − Silica membrane becomes negatively charges, like the
Conditions: DNA, so they repel & DNA gets coated in water & comes out in the solution
 Gel loading buffer ● 0.05% bromophenol blue
B. RNA Extraction Components: ● 40% sucrose
● 0.1 M EDTA
● RNAs are single-stranded and are more fragile than DNA ● 0.5% SDS
● RNAs are easily damaged by RNAses (found in different sources such as skin)
● It is useful to allocate an RNAse free area of the laboratory 1. We first prepare the agarose, before it solidifies we put comb because we want them to have holes.
● More sensitive method In the holes, we put the samples, either DNA extracts of product of the PCR.
● Cold chain transport 2. We cover the entire set up with buffer up to the part where it covers the gel.
● Same step in DNA 3. We then use a gel loading buffer to make the solution heavy or denser due to the 40% sucrose. If we
● We extract RNA for causing infectious diseases and the positive agent is not a DNA virus (sars-cov2) don’t put a loading buffer, DNA will not go to the well.
4. Using a pipette we place it over the well and release it the sample will go down
5. Then we now turn on the electricity. Sample will go the cathode. DNA moves towards anode. After
Lysis ● Uses detergents or phenol in high salt concentration and Guanidine
gel electrophoresis
isothiocyanate
6. DNA will be stained using Ethidium bromide or HiSyBr Green. We then put it in the UV to see the
bands of the DNA
Binding Agent ● Silica 7. those that travel farther to the well means they are smaller fragments, those that travel closer are
● Might have column or magnetic beams larger fragments

Quantifying DNA/RNA

● Use spectrophotometer to quantify

● Wavelength = 260 nm
● DNA–1 OD= 50 ug/ml
● RNA–1 OD=40 ug/ml
● DNA concentration = absorbance x 50 ug/ml x dilution factor

Example: A DNA prep diluted 1:100 has an absorbance reading of 0.300 at 260 nm.

DNA concentration = 0.300 x 50 ug/ml x 100 = 1,500 ug/ml

DNA concentration systems

● Nanodrop - 1 or 2 um
● Quibit system - fluorescent dyes

Visualizing DNA

Agarose Gel ● Run the DNA in an agarose gel

Electrophoresis ● Agarose gel have 2 ends
○ Negative - where the DNA sample is placed
○ Positive - since the DNA is negative it is more attracted to the positive end
● DNA fragments can move fast in the agarose gel.

Gel 2.5% agarose depending on your need

Dissolve it at 100% buffer

Buffers: ● TBE
● TAE

DNA ladder 100 bp DNA ladder or 1 kb DNA ladder