DNA ISOLATION FROM BLOOD
Content
Introduction .............................................................................................................................................2
Materials for DNA Isolation .....................................................................................................................2
Method for DNA Isolation .......................................................................................................................3
Using Spin Column ..................................................................................................................................4
Ready to Use DNA ...................................................................................................................................6
1
DNA ISOLATION FROM BLOOD
Introduction
DNA isolation is required for genetic analysis, which is helpful in scientific, medical, or forensic
fields. However; to carry out any process in molecular biology they require an initial pure sample of
DNA. DNA purification and DNA extraction involve separating the DNA present in the nucleus of the
cell from other cellular components and protecting it from degradation by cellular enzymes.
Materials for DNA Isolation
 Gloves
 Tissue sample
 Forceps/scissors
 Pipettes and tips
 1.5 mL microcentrifuge tube (2)
 Buffer ATL (Lysis buffer)
 Proteinase K
 Buffer AL (contains chaotropic salt, an irritant)
 Ethanol (96%-100%)
 DNeasy mini-column2
 Buffer AW1 (also contains chaotropic salt)
 Buffer AW2 (contains sodium azide)
 2 mL collection tube
 Microcentrifuges
 Water bath
 Vortex
2
DNA ISOLATION FROM BLOOD
Method for DNA Isolation
1. Add Proteinase K in a 2.0 mL microcentrifuge tube.
a. It is commonly used to digest and inactivate DNase and RNase during in nucleic acid
purification.
2. Blood sample that will be used for DNA isolation is added in a 2.0 mL microcentrifuge tube.
3. Add Buffer AL (Lysis buffer).
a.
A
lysis
buffer
is
the
action
of
cells membrane.
3
a
specific
Lysine
that
disrupts
the
DNA ISOLATION FROM BLOOD
4. Mix thoroughly by vortexing, and incubate at 56°C for 10 min.
a. Longer incubation times have no effect on yield or quality of the purified DNA.
5. Adding 96% ethanol to lysate after incubation.
a. It will aggregate together, giving a pellet upon centrifugation. If enough ethanol is added
electrical attraction between phosphate groups and any positive ions present in solution
becomes strong enough to form stable ionic bonds and precipitate DNA.
6. Performing 15 seconds vortex for mixing.
7. Spin column placed in a 2 ml collection tube and added the mixture to column of commercial kit.
8. Centrifuge at 6000 x g (8000 rpm) for 1 minute.
9. Discard flow-through and collection tube. Place the spin column in a new 2 ml collection tube.
4
DNA ISOLATION FROM BLOOD
10. Add Buffer AW1, and centrifuge for 1 minute at 6000 x g (8000 rpm).
a. Buffer AW1 contains Guanidinium Chloride (guanidine hydrochloride). This is used to
denature proteins in the sample. They will then flow through the column and will be discarded
with the wash.
b. Buffer AW1 and Buffer AW2 are supplied as concentrates.Before using for the first time,
add the appropriate volume of ethanol (96–100%) as indicated on the bottle and shake
thoroughly.
11. Discard flow-through and collection tube. Place the spin column in a new 2 ml collection tube.
12. Add Buffer AW2, and centrifuge for 4 minute at 20000 x g (14000 rpm).
a. Buffer AW2 is essentially 70% EtOH. 30 mls of 100% EtOH is added to the 13 mls of
"concentrate" included in the bottle. 70% EtOH is used to remove salts from your column and
aid in purifying the DNA.
13. Transferring the column to in a clean 1.5 ml or 2 ml microcentrifuge tube.
5
DNA ISOLATION FROM BLOOD
14. Pipet Buffer AE (elution buffer) directly onto the membrane and centrifuge for 1 min at 6000 x
g (8000 rpm) to elute.
a. Buffer AE is 10 mM Tris·Cl, 0.5 mM EDTA, pH 9.0. Elution with Buffer AE
guaranteesoptimal recovery and stability of eluted DNA.
15. After centrifuge ready to use or store the DNA at -20 °C.
6