Genomic DNA isolation from Goat Blood

using the Phenol: Chloroform: Isoamyl

alcohol (PCI) method

Aim: To isolate genomic DNA from Goat Blood sample using PCI method

Principle: DNA is the prerequisite for life’s inception that transfers hereditary information.
Isolation of genomic DNA is a most important step in a variety of clinically related studies
including genetics, genomics, gene polymorphism, DNA fingerprinting and gene sequencing.
Whole blood is one of the accessible sources to obtain genomic DNA.
Phenol: Chloroform: Isoamyl alcohol (PCI) method is one of the conventional methods which is
used to isolate genomic DNA. Phenol-chloroform isoamyl alcohol relies on the principle of liquid-
liquid extraction of biomolecules. It denatures the protein portion of a cell and removes it
followed by separating genomic DNA into a soluble phase.
1. Phenol is used for it to unfold the protein structure and digest it as DNA is insoluble in
phenol because phenol is a nonpolar solution.
2. Chloroform increases the efficiency of phenol to denature the protein. Here, chloroform
allows proper separation of the organic phase and aqueous phase and keeps DNA
protected into the aqueous phase.
3. Isoamyl alcohol helps in reducing foaming between interphases. It prevents the
emulsification of a solution. The liquid phase contains DNA and the organic phase
contains lipid, proteins and other impurities. The anti-foaming agent, isoamyl alcohol
stabilizes the interphase by removing the foaming and increases the purity of DNA.

Material Required: K2 EDTA-vacutainer, falcon tubes, Centrifuge, Ice box, Micropipette

Reagents/Chemicals:
a) RBC Lysis Buffer: Sucrose-1M Tris-HCl (pH-8) + 1M MgCl2 + Triton X-100
b) WBC Lysis Buffer: 1M Tris-HCl (pH-8) + 0.5M Na2-EDTA
c) TE buffer: 10 mM Tris-HCl + 1mM EDTA (pH-8)
d) Chilled double distilled water
e) 1M and 5M NaCl2
f) 20% SDS detergent
g) proteinase-K
h) PCI (Phenol:Chloroform:Isoamyl alcohol) of ratio (25:24:1)
i) Chloroform: Isoamyl alcohol (24:1)
j) Chilled 3.5M sodium acetate (pH 5.2)
k) Chilled absolute (100%) and 70% alcohol
Procedure:
1. Clotted sample is thawed rapidly in a 37°C water bath to liquefy the blood samples.
2. 5ml blood sample is collected in K2 EDTA-vacutainer (K2 EDTA is a type of
anticoagulant used in the blood collections tubes to prevent blood coagulation during
storage by chelating calcium ions required by the coagulation process)
3. The whole blood is transferred into a 15 ml falcon tube and centrifuged for 20 min at
3500 rpm to separate the buffy coat.
4. The separated buffy coat is then transferred to a 15ml falcon tube into which 1.5 ml of
RBC lysis buffer is added.
5. Chilled double distilled water (3ml) is added to this mixture and centrifuged at 3500 rpm
for 12 minutes.
6. The pellet is collected and suspended in equal volumes of RBC lysis buffer and chilled
distilled water.
7. The pellet is made uniform and then centrifuged again at 3500 rpm for 12 min. This step
is repeated until a clear WBC pellet is obtained.
8. 1ml of WBC lysis buffer is added to the RBC-free pellet.
9. To this mixture, 1M NaCl2 is added along with 250 µl of 20% detergent SDS (Sigma,
USA) and proteinase-K (20mg/µl) (Sigma, USA) and incubated for 10hrs at 37ºC.
10. After incubation, 200 µl 5M NaCl2 is added and Centrifuged at 10000 rpm for 10 min at
4ºC.
11. The supernatant is mixed with an equal volume of PCI mixture.
12. The tubes are mixed well and centrifuged at 8000 rpm for 5 min at 4ºC.
13. The supernatant is transferred into a centrifuge tube and equal volume of Chloroform:
Isoamyl alcohol (24:1) is added and centrifuged at 8000 rpm for 5 min at 4ºC.
14. DNA is precipitated by the addition of 200µl of chilled 3.5M sodium acetate (pH 5.2)
(Sigma, USA) and -20ºC chilled absolute (100%) alcohol to the obtained supernatant.
15. The precipitate is washed with chilled 70% alcohol twice to remove contaminants.
16. The DNA is precipitated and transferred into a 1.5 ml fresh tube and the pellet is air dried
at 55º C for 10 min.
17. Genomic DNA precipitant is re-suspended in 150 µl of TE buffer

Flow Chart:
Thaw Clotted sample in a 37°C water bath
↓
Collect 5ml blood in K2 EDTA-vacutainer
↓
Transfer whole blood into a 15 ml falcon tube and centrifuge for 20 min at 3500 rpm
↓
Transfer separated buffy coat to a 15ml falcon tube and add 1.5 ml of RBC lysis buffer
↓
Add Chilled double distilled water (3ml) and centrifuge at 3500 rpm for 12 minutes
↓
 suspended in equal volumes of RBC lysis buffer and chilled distilled water and
centrifuge at 3500 rpm for 12 min, repeat until a clear WBC pellet is obtained
↓
add 1ml of WBC lysis buffer to the RBC-free pellet, add 1M NaCl2 along with 250 µl of 20%
detergent SDS and proteinase-K (20mg/µl), and
incubate for 10hrs at 37ºC
↓
add 200 µl 5M NaCl Centrifuge at 10000 rpm for 10 min at 4ºC
↓
Add equal volume of PCI mixture to the supernatant, mix well centrifuge at 8000 rpm for 5 min
at4ºC
↓
centrifuged at 8000 rpm for 5 min at 4ºC
↓
Transfer the supernatant into a centrifuge tube, add equal volume of Chloroform: Isoamyl
alcohol (24:1), centrifuge at 8000 rpm for 5 min at 4ºC
↓
Add 200µl of chilled 3.5M sodium acetate (pH 5.2) (Sigma, USA) and -20ºC chilled absolute
(100%) alcohol to the obtained supernatant
↓
Wash precipitate with chilled 70% alcohol twice to remove contaminants
↓
Transfer precipitated DNA into a 1.5 ml fresh tube and air dry at 55º C for 10 min
↓
Resuspend Genomic DNA precipitant in 150 µl of TE buffer

Storage:
The DNA Sample can be labeled and stored in -20ºC freezer for upto 7 years for further
molecular studies.

Other ways-
1. at –196°C (storage in liquid nitrogen)
2. at –80°C as a precipitate under ethanol
3. preserved by keeping it at Room temperature on a dry, solid matrix (for short durations)

To evaluate the probability of DNA integrity, quality of DNA isolated is assessed by agarose gel
electrophoresis. 10 μl of extracted gDNA along with bromophenol blue dye (Sigma, USA) is
loaded on 0.8% agarose gel and subjected to electrophoresis (Genetixs Biotech, Asia). The
bands are visualized by UV-transillumination gel documentation system (Bio-Rad XR Imager,
USA).

Gel Electrophoresis

Principle: Electrophoresis is the process of separating and characterizing molecules based on

their size and charge. The negatively charged DNA molecules migrate towards the positive
charge under the influence of constant current, thus the separation depends on the mass and
charge of DNA. The DNA molecules are forced to move through the agarose gel pores. The rate
of the migration depends on-

1. strength of the field

2. hydrophobicity of the DNA
3. ionic strength of the buffer
4. size and shape of the DNA
5. temperature of the buffer

Three of the important factors that affect DNA migration rate are:

1. Agarose concentration
If the concentration of agarose is lower, the DNA can migrate easily and faster and vice
versa.
2. Confirmation of DNA
The supercoiled DNA migrates faster than the linear DNA and circular DNA. The
supercoiled form of DNA is more compact than other forms causing speedy migration.
3. Size of DNA
Larger DNA fragments run slower while smaller DNA fragments run faster. Shorter PCR
amplicons migrate faster than the gDNA.
Graphical representation of the electrophoresis apparatus.

Comb setup and migration of DNA in a gel.

Material Required: Gel caster, electrophoresis chamber, voltage source, gel tray, comb, oven,
UV transilluminator, pipettes, tips, flask, weight balance

Reagents/Chemicals:
a) Agarose powder
b) TAE buffer
For 250 ml of 10X TAE buffer, add
- 400mM Tris
- 200mM acetic acid
- 10mM EDTA
- Distilled Water to make the final volume of 250ml
To make 1x TAE from 10X TAE stock, dilute 10ml of stock into 90 ml of distilled
water.
c) Ethidium bromide (EtBr)
d) Bromophenol blue dye

Procedure:
1. Prepare 0.8% Agar Gel by dissolving 0.8 gm of agar powder in 100ml of TAE buffer.
Heat for 3-5 minutes till the solution becomes clear which indicates that the agarose
powder is dissolved properly into the buffer.
2. Add 0.5μg/ml EtBr and shake well to mix it properly, (shake it gently so that it can’t form
bubbles).
3. Place the gel tray on a gel caster and tighten it with the help of clamps. Put the gel comb
on the gel tray and pour the gel into the gel caster and wait until it becomes solid.
4. Place the gel tray into the chamber and fill the electrophoresis chamber with the TAE
buffer till the gel is fully submerged.
5. Now mix 10μl of extracted genomic DNA sample along with bromophenol blue dye and
load into the well. Also add the DNA ladder in a separate well.
6. Connect the electrodes to the power pack and run the gel at 100v for 45 to 60min. Run
the samples until the DNA covers a 75% distance towards the positive node.
7. Turn off the power supply, carefully remove the gel from the electrophoresis chamber,
and drain off the buffer.
8. Now place the gel carefully on the UV transilluminator platform and close the lead of the
transilluminator.

Results:
 Agarose gel electrophoresis of genomic DNA obtained from uncleaved goat blood sample

Reference:
1. Goud, T. S., Upadhyay, R. C., Kumar, A., Karri, S., Choudhary, R., Ashraf, S., ... & Kiranmai, C.
(2018). Novel extraction of high quality genomic DNA from frozen bovine blood samples by using
detergent method. Open veterinary journal, 8(4), 415-422.
2. Kumar, O. S., Sharma, M. K., & Singh, D. (2006). DNA isolation from goat blood using different
brands of household detergents and its downstream application.
3. Yaradoddi, Jayachandra. (2013). Re: Best way to preserve isolated DNA?. Retrieved from:
https://www.researchgate.net/post/Best_way_to_preserve_isolated_DNA/
51d6a688d039b132069c1f30/citation/download.
4. Brown, T. A. (2016). Gene cloning and DNA analysis: an introduction. John Wiley & Sons.
5. https://geneticeducation.co.in/phenol-chloroform-dna-extraction-basics-preparation-of-chemicals-
and-protocol/
6. https://geneticeducation.co.in/agarose-gel-electrophoresis-definition-principle-process-protocol-
and-applications/