Download Revision BIOC 432 LAB

Lecturer Bahiya Osrah
2 nd Term 2012
DNA isolation Lab
DNA isolation from strawberries
See the attached File on my website
Why we chose strawberries:
It is a good candidate for demonstrating DNA extraction since it is octoploid with eight copies of each gene in the strawberry genome.
Explanation
The detergent in the shampoo helps to dissolve the phospholipid bilayers of the
cell membrane and organelles. The salt helps keep the proteins in the extract
layer so they aren’t precipitated with the DNA.
DNA is not soluble in ethanol. When molecules are soluble, they are dispersed in
the solution and are therefore not visible. When molecules are insoluble, they
clump together and become visible. The colder the ethanol, the less soluble the
DNA will be in it yielding more visible “clumping.” This is why it is important for
the ethanol to be kept in a freezer or ice bath.
Isolation of DNA from Tissues:
To extract DNA from tissue/cells of interest, the cells have to be separated and
the cell membranes have to be disrupted.
Reagents : Lysing step
1.
EDTA (Ethylenediaminetetraacetate) which removes Mg+2 and Ca+2 ions
These ions are essential for preserving the overall structure of the cell
membrane
To inhibit Dnase ( Mg+2 and Ca+2 are cofactors)
2.
SDS (Sodiumdodecylsulfate), which aids in disrupting the cell membranes
by removing the lipids of the cell membranes, and solubilized the proteins
both are included in the extraction buffer which lysing the cells,
3.
Protein enzyme or protease can be used to break down proteins.
After the lysing step and centrifugation the cell
extract now contain:
DNA , protein and RNA.
to remove these contaminants, leaving the DNA in a pure form we need to
add:
1.Phenol:
The standard way to de-proteinize (precipitate protein) a cell extract is to
add phenol or a 111 mixture of phenol1 chloroform.
These organic solvents precipitate proteins but leave the nucleic acids in
aqueous solutions.The aqueous solution of nucleic acid can be removed with
a pipette.
2. Ribonuclease enzyme: Ribonuclease enzyme to degrade RNA.
Don’t forget to know
 Isolation of DNA from spleen
Have a look on the protocol of the isolation of DNA from spleen
 STUDY:
The reagents and their purposes:
Reagents:
The tissue is homogenized in isotonic saline buffered with sodium citrate PH7.4:
Because at this ionic strength, the deoxyribonucleoprotein is insoluble and separates well
from other proteins.
The sodium citrate or EDTA (Ethylenediaminetetraacetate) is used to:
Inhibit deoxyribonuclease activity by binding Ca++ and Mg++, which are cofactors for this
enzyme.
Some DNA isolation protocols used SDS (sodiumdodecylsulfate) to:
Disturb the cell membranes by removing thr lipids and solubilized the protein.
Don’t forget to know
Proteinase is used to:
Break down the yield protein during the isolation process.
The extraction procedure is carried out under cold condition:
so that any residual DNA'ase activity is minimal.
ice-cold ethanol:
The DNA is finally precipitated as a fibrous white mass by the addition of
ethanol.
DNA less soluble in cold ethanol.
Structure of RNA
 RNA exist as a single strand.
 Ribose Sugar (5 carbon sugar)
 Phosphate group
 Adenine, Uracil, Cytosine, Guanine
 For RNA, nucleosides are formed similarly to DNA.
 Hairpin is a common secondary/tertiary structure.
 Double stranded RNA can also exists in mammals
RNA Types
New types of RNA
snRNA - Small nuclear RNA
•
is a class of small RNA molecules that are found within the nucleus of
eukaryotic cells.
•
They are involved in a variety of important processes such as RNA
splicing (removal of introns), regulation of transcription factors and
maintaining the telomeres.
snoRNA - Small nucleolar RNA
•
snoRNAs are involved in rRNA modification.
•
They are located in the nucleolus .
Other RNA’s involved in Gene Expression Regulation:
siRNA- RNA interference
miRNAs- Micro RNA
siRNA
(Gene Silencing)
Degrades mRNA then
it inactivates the gene
translation
miRNAs
• Tiny 21–24-nucleotide RNAs
• Non coding small RNAs
• unlike siRNAs, miRNAs downregulate expression
after translation initiation without affecting mRNA
stability
• stem-loop structure is highly Conserved
Translation
Inhibition
TBE buffer
Tris-Borate:
To allow the current conductivity
EDTA (Ethylenediaminetetraacetate):
Divalent cations chelating agents, which inhibits subsequent
enzymatic reactions.
Note:
The TBE should not be very concentrated:Because that would
make the current very high and a lot of heat would be
generated which could cause gel melting and DNA
denaturation.
DON’T forget::
The Bromophenol Blue Dye:
1. blue tracking dye that migrate usually as 300 bp DNA
2. This allows the samples to be seen when loading onto
the gel
3. increases the density of the samples, causing them to
sink into the gel wells
DNA ladder
To identify the separated or migrated DNA bands sizes
in the gel
Ethidium bromide
Interchelates the DNA bases and glows under the UV light
to give the pink color which allows the DNA visuilization
The differences between Agarose and
polyacryamide
Agarose:
1. It is a polysaccharide extracted from seaweed
2. It is typically used at concentrations of 0.5% to 3%.
3. Its preparation is easier than polyacrylamide
4. Agarose gels have a large range of separation, but relatively low
resolving power.
5. DNA fragments from about 0.2 kbp to 50 kbp can be separated
in agarose.
Polyacrylamide:
1. It is a cross-linked polymer of acrylamide.
2. A wide range of conc. can be used between 3.5% to 20%
(Advantage to give higher resolution to separate very small DNA
fragments that are differ in a one bp and so used for DNA
sequencing).
3. Polyacrylamide gels are more annoying to prepare than agarose
gels and toxic (Disadvantage). Because oxygen inhibits the
polymerization process, they must be poured between glass
plates (or cylinders).
4. Polyacrylamide gels have a small range of separation, but very
high resolving power.
5. DNA less than 0.5 kbp can be separated by polyacrylamide.
SDS Gel Electrophoresis
SDS-PAGE
For protein separation
In SDS separations, migration is determined not by
intrinsic electrical charge of polypeptides but by
molecular weight
Sodium dodecylsulfate (SDS) is an anionic
detergent
SDS confers a net negative charge to the polypeptide in proportion to its
length
denatures secondary and non–disulfide–linked tertiary
structures by wrapping around the polypeptide backbone.
SDS Gel Electrophoresis
Finally
Identify the separated DNA band
sizes
DNA Ladder
 12,000 bp
 5,000
Note: bromophenol
blue migrates at
approximately the
same rate as a 300 bp
DNA molecule
DNA
migration
bromophenol blue
+
 2,000
 1,650
 1,000
 850
 650
 500
 400
 300
 200
 100
Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy to determine
the sizes of unknown DNAs.
Bioinformatics
All the information about the sequence of human genome can be obtained
from large databases such as
• NCBI (National Center for Biotechnology Information)
• DDBJ (DNA Data Bank of Japan)
• EMBL (European Molecular Biology Laboratory)
• Protein structure databases
PDB =Protein data bank
• PubMed
Literature references
• Nucleotide sequence databases
NCBI (GeneBank)