The document outlines the process of DNA extraction using various reagents and buffers, highlighting the roles of each component such as Tris-HCl, EDTA, NaOH, and SDS in separating plasmid DNA from chromosomal DNA. It describes the importance of maintaining optimal pH levels and the effects of different agents on DNA structure during extraction. Additionally, it details the formation of precipitates and how gel electrophoresis can be used to analyze the integrity of plasmid and chromosomal DNA post-extraction.

1. Extraction kits – reagents and functions
How do kits work on DNA
1) Centrifugation results in the separation between cells and the
liquid medium they are sitting in. Afterwards, Tris-HCl and EDTA
enter the process: The buffer offers the right pH (=7.4) for the
subsequent reaction to take place, and the EDTA chelates (bonds)
1
with metal ions (EDTA has great affinity with metal ions; and Mg-
ion present in DNase is a cofactor responsible for DNase
denaturing DNA).
2) A second step will bring NaOH and SDS to the equation:
Sodium hydroxide will relax the double strand and allow the
melting of the double chain into a single stranded chain (due to
an increase in the pH  14). SDS is an anionic detergent which
disrupts the cell membrane and destabilises all hydrophobic
interactions holding macromolecules in their native form.
Thus, SDS disrupts the membrane phospholipidic bilayer allowing
the intracellular material to abandon the cell.
2

2. Extraction kits – reagents and functions
How do kits work on DNA
3) A third solution will be formed after adding Potassium 3
Acetate (KOAc) to the previous mixture. KOAc will lower
the pH levels to approximately 4 making the solution acidic
(optimal for the DNA molecule). The rationale for using
KOAc is that chromosomal DNA is found in a double-
stranded supercoiled fashion whereas plasmidic DNA
entails a circular structure; when one breaks the bacterial
DNA molecule it will be almost impossible for the
reannealing to happen. On the other hand that is not quite
the case with the plasmidic DNA for the length of the
molecule is short, thus allowing complementary bases to
find their matches more easily.
4) This is where a chromosomal precipitate will be
generated and subsequently discarded, whilst plasmidic
DNA will be squirted onto the column filter for further
elution with either buffer or distilled water. Long story 4
short, genomic DNA produces a macroscopic clew and
plasmidic DNA reanneals into its circular original shape.

3. Extraction kits – reagents and functions
How do kits work on DNA
A digested plasmid, say by EcoRI, will produce a
linear chain that when ran in a agarose gel yields a
typical band with the same length as the original
plasmid circular structure. However, an
unsuccessfully uncut plasmid can produce 3 types
Nicked
of bands:
Open
Circular
• Band 1 – a band of DNA with average mobility
corresponding to a linear fragment of DNA.
• Band 2 – a completely uncut plasmid that in an Plasmid
agarose gel yields a fast travelling fragment DNA
resulting in a band further south on the gel bed. digested Fully
• Band 3 – heavy fragment, thus by EcoRI uncut
slower, corresponding to a nicked plasmid
where only one of the two chains is disrupted.

4. Biomolecular kits and their reagents
QIAprep miniprep handbook
P1 buffer
1M Tris HCl pH 8.0 2.5 ml
0.5 M EDTA pH 8.0 1 ml
10 mg/ml RNAseA, boiled 0.5 ml
dH2O 46 ml
50ml total
Tris/HCl - absorb counter ions (+H and -OH) so as to help keep the solution at a stable pH level.
EDTA - acts as a strong chelating agent (forms coordinate bonds with metals) holding metals in a non-ionisable form.
P2 buffer
2M NaOH 5 ml
20% SDS 2.5 ml
dH2O 42.5 ml
50 ml total
NaOH – To separate the bacterial chromosomal DNA and sheared (cut off) DNA from plasmid DNA, sodium hydroxide is often used. When the solution is basic, for example, when sodium
hydroxide is added, double-stranded DNA molecules denature and separate.
SDS - used as a lysing solution because, as a detergent it dissolves the cell membrane which is made out of lipids, but is also used to denature proteins once the cell has been lysed.
Buffer N3 contains:
3.0 M KOAc, pH 5.5 (29.5 g/ 100ml water)
Acetic acid – is used to help separate plasmid DNA from chromosomal DNA, Single strands of linear DNA are insoluble in high salt. They will precipitate out, forming a solid. Adding acetic acid to
SDS detergent solutions forms solids of cellular debris as well as denatured chromosomal linear DNA. Circular plasmid DNA is not insoluble in high salt. Plasmid DNA will remain in solution, thus
separating the desired plasmid DNA from the rest of the DNA in the cell.
Buffer PB:
Guanidine hydrochloride - denaturing agent that break up non-covalent bonds like those that stabilize a protein's conformation and are often added to denaturation solutions.
Isopropanol - is used to precipitate the plasmid DNA, DNA is insoluble in alcohol and clumps or clings together. Centrifuging will cause the precipitate to form a pellet which can be decanted
from the unwanted supernatant.
Buffer PE
75% EtOH - used to precipitate the plasmid DNA
25 mM NaCl, - salt used to precipitate the plasmid DNA
5 mM Tris-Hcl, pH 7.5
Buffer EB
10 mM Tris·Cl, pH 8-8.5
Rnase A - a small (124 amino acids) digestive enzyme secreted by the pancreas and is an endonuclease specifically designed to hydrolyze RNA (but not DNA) phosphodiester bonds which
covalently link ribonucleotides particularly those linked to pyrimidine bases such as uracil. Cleaves either single-stranded or double-stranded RNA.