DNA Isolation
Novi Silvia Hardiany
Dept.Biochemistry & Molecular
Biology FMUI
1. DNA Isolation (Chelex method)
• Extract DNA from students cheek cells
• Procedure:
v Pour the saline into your mouth and rinse vigorously for 30
seconds
v Transfer 1 ml of your saline rinse into the microtest tube
(NOT the screwcap tube)
v Spin your tube in a balanced centrifuge at full speed for 2
minutes
v After pelleting your cells, pour off the saline
v Resuspend the pellet by vortexing or flicking the tube so that no clumps of cells
remain
v Transfer 20 μl of your resuspended cells to the screwcap tube containing
InstaGene
v Screw the cap tightly on the tube. Shake or vortex to mix the tube contents.
v Place the tubes in the foam micro test-tube holder, and incubate at 56°C for 10
minutes in a water bath. At the halfway point (5 minutes), shake or vortex the
tubes gently, then place back in the 56°C water bath for the remaining 5 minutes.
v Remove the tubes, shake or vortex, and place the tubes in a boiling water bath
(100°C). Incubate at 100°C for 5 minutes.
v Remove the tubes from the boiling water bath and shake or vortex the contents to
resuspend. Pellet the matrix by spinning at 6,000 x g for 5 minutes in a centrifuge.
v Read the absorbance of your DNA at 260 nm and 280 nm
Add 10 µL of DNA + 990 µL aquabidest (100 x dilution)
DNA Concentration = A 260 nm x 50 µg/mL x 100 (Dilution factor)
Purity of DNA = A 260 nm/ A 280 nm
• InstaGene™ matrix consist of a suspension
negatively charged Chelex® microscopic bead
→ binds cellular magnesium ions
•56°C - loosens connective tissue, soften plasma
membrane, release clump of cells from each
other & inactivates DNases
• 100°C - ruptures cell membranes, release DNA
from nuclei and denatures proteins (DNases)
Cell membrane
Nuclear membrane
Mg++
Genomic DNA
InstaGene
Extraction Mg++
Mg++
Mg++
Heat disrupts membranes Mg++
InstaGene matrix binds
Mg++ released cellular Mg++
Bahan
• Kit Wizard Genomic Isolation (Promega):
- Cell Lysis Solution
- Nuclei Lysis Solution
- RNase solution (optional)
- Protein precipitation solution
- Isopropanol
- Etanol 70 %
- DNA rehydration solution
• Whole Blood
Prosedur (1)
• 300 μL whole blood ditambahkan pada tabung
1.5 mL yang sudah berisi 900 μL cell lysis
solution
• Campur tabung dengan dibalik2 sebanyak 5-6
kali
• Inkubasi 10 menit
Prosedur (2)
• Buang supernatan, sisakan sebanyak 10-20 μL
pellet putih vortex
• Tambahkan 300 μL Nuclei lysis solution
campur 5-6 kali
• Tambahkan 100 μL protein precipitation
solution vortex selama 10-20 detik
Prosedur (3)
• Sentrifugasi pada 13.000 – 16.000 xg selama 3
menit pada suhu ruang pellet coklat
• Pindahkan supernatan pada tabung 1.5 mL yang
sudah berisi 300 μL isopropanol bolak balikkan
tabung beberapa kali benang halus putih
• Sentrifugasi pada 13.000 – 16.000 xg selama 1
menit buang supernatan pellet putih
Prosedur (4)
• Tambahkan 300 μL etanol 70 % balik2
tabung sentrifugasi buang supernatan
pellet putih
• Tambahkan 100 μL DNA Rehydration solution
inkubasi overnight pada suhu ruang atau
4◦C
Pengukuran Kadar DNA
• Konsentrasi DNA spektrofotometri
= A260 nm x 50 µg/mL x faktor pengenceran
• Kemurnian DNA = A260/A280
Purity Index: 1.75 – 2.0
2. PCR
• What Is Needed for PCR?
1. Template (the DNA you want to amplify for the study)
2. Sequence-specific primers flanking the target sequence:
5’ 3’ 5’ 3’
Forward primer
Reverse primer
3’ 5’
5’ 3’
Target sequence
3. Nucleotides (dATP, dCTP, dGTP, dTTP)
4. Magnesium ions (enzyme cofactor)
5. Buffer, containing salt
6. Taq polymerase
The Target Sequence
§ PV92 Alu insertion
§ Located on Chromosome 16
§ A member of Alu repeat family
§ Human-specific Alu insertion
§ Found in a non-coding region of your DNA
§ Not diagnostic for any disease or disorder
PCR Step
PCR Results
• The PV92 Alu is
dimorphic so there
are two possible
PCR products: 641 300 bp Alu insert
bp and 941 bp
Actual Alu PCR
Results
Procedure
1. Label a PCR tube and a capless micro test tube with your
initials, place the PCR tube in the capless tube and place
both in the foam holder.
2. Transfer 20 μl of the DNA template from the tube into the
PCR tube containing 20 µl of Master Mix (yellow colour).
3. Mix by pipetting up and down 2–3 times. Cap
the PCR tube tightly.
4. Place the PCR tube into the thermal cycler.
The reactions will undergo 40 cycles of PCR
amplification.
Electrophoresis of PCR product will be done
in the next practice
