Genomic DNA extraction by spin column method
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One of the protocols for DNA extraction by spin column method. Slight variation may be there in different workplaces.
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Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 3
Equipment
1) EDTA vial
2) Spin columns with collecting tube
3) Microcentrifuge tube
4) Dry incubator
5) Vortex mixer
6) Microcentrifuge machine
7) Micropipettes (adjustible)
Requisites for Gel Electrophoresis
8) Gel casting tray
9) Well combs
10) Electrophoresis tank
11) UV transilluminator
Reagents
Reagents
1. Sample- Whole blood collected in EDTA vial
2. Protease k
3. Lysis buffer (Tris-HCl (0.5-50mM)+ EDTA+ SDS
(0.5-2%) or Triton X-100+ NaCl (50-200mM))
4. Wash buffer AW1 (Low-Salt or Chaotropic Wash)
[Guanidine HCl or GuSCN (0.5–1 M)+ Tris-HCl
(10 mM, pH 7.5–8.0)+ Ethanol (20–40%)]
5. Wash buffer AW2 (Ethanol (70–80%)+ Tris-HCl or
TE Buffer (optional, low concentration))
6. Ethanol (96-100%)
7. Elution buffer (10 mM Tris-HCl (pH 8.0))
For electrophoresis
1) Agarose powder (Molecular grade) 2%
2) TAE buffer-pH 8.0
3) Ethidium bromide- Stock concentration of 10mg/ml
4) Tagging dye- bromophenol blue](https://image.slidesharecdn.com/dnaextraction-250726171916-8e401145/85/Genomic-DNA-extraction-by-spin-column-method-3-320.jpg)
Genomic DNA extraction by spin column method
- 1. 1 Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 1 DNA extraction Dr V P Acharya MD, PhD
- 2. 2 Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 2 Extraction of genomic DNA by Spin column method
- 3. 3 Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 3 Equipment 1) EDTA vial 2) Spin columns with collecting tube 3) Microcentrifuge tube 4) Dry incubator 5) Vortex mixer 6) Microcentrifuge machine 7) Micropipettes (adjustible) Requisites for Gel Electrophoresis 8) Gel casting tray 9) Well combs 10) Electrophoresis tank 11) UV transilluminator Reagents Reagents 1. Sample- Whole blood collected in EDTA vial 2. Protease k 3. Lysis buffer (Tris-HCl (0.5-50mM)+ EDTA+ SDS (0.5-2%) or Triton X-100+ NaCl (50-200mM)) 4. Wash buffer AW1 (Low-Salt or Chaotropic Wash) [Guanidine HCl or GuSCN (0.5–1 M)+ Tris-HCl (10 mM, pH 7.5–8.0)+ Ethanol (20–40%)] 5. Wash buffer AW2 (Ethanol (70–80%)+ Tris-HCl or TE Buffer (optional, low concentration)) 6. Ethanol (96-100%) 7. Elution buffer (10 mM Tris-HCl (pH 8.0)) For electrophoresis 1) Agarose powder (Molecular grade) 2% 2) TAE buffer-pH 8.0 3) Ethidium bromide- Stock concentration of 10mg/ml 4) Tagging dye- bromophenol blue
- 4. 4 Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 4 Sample EDTA whole blood
- 5. 5 Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 5 1.Pipet 20μl of QUIAGEN Protease K into a 1.5 ml microcentrifuge tube. Add 200 μl of whole blood sample
- 6. 6 Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 6 2. Add 200 μl lysis buffer. Mix thoroughly by vortex mixer
- 7. 7 Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 7 3. Incubate at 560 c for 10 minutes. Briefly centrifuge to remove drops from the lid
- 8. 8 Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 8 4. Add 200 μl of ethanol (96-100%) and mix thoroughly by vortexing. Briefly centrifuge to remove droplets from the lid.
- 9. 9 Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 9 5. Pipet the mixture onto QIAamp mini spin column (2ml collection tube) and centrifuge at 8000rpm for 1 minute. Discard the flow through and collection tube.
- 10. 10 Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 10 6. Place the QIAamp mini spin column in a new 2ml collection tube and add 500μl of wash buffer AW1. Centrifuge at 8000rpm x 1 min. Discard the flow through and collection tube.
- 11. 11 Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 11 7. Place the QIAamp mini spin column in a new 2ml collection tube and add 500μl of wash buffer AW2. Centrifuge at 14000rpm x 3 min. Discard the flow through and collection tube.
- 12. 12 Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 12 8. Recommended: Place the QIAamp mini spin column in a 2ml collection tube (normal microcentrifuge tube) and centrifuge at full speed for 1 min. This eliminates the chance of possible buffer AW2 carryover.
- 13. 13 Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 13 9. Place the QIAamp mini spin column in a new 1.5 ml microcentrifuge tube, add 100/200 μl elution buffer AE and incubate at room temperature (15-250 c) for 1 min. Centrifuge at 8000rpm for 1 min to elute the DNA.
- 14. 14 Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 14 10. To know how much DNA is extracted, it is run on Gel electrophoresis and compared with ladder sequence.
- 15. 15 Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 15 The Run
- 16. 16 Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 16 Agarose gel electrophoresis 1. 2gm of agarose powder was mixed with 100 ml of 1x TAE buffer and warmed in the microwave oven for 1-3 minutes till it was completely dissolved. 2. The agarose gel was allowed to cool down to 500 c and then 2-3 μl of ethidium bromide (0.2-0.5 μg/ml) was added. EtBr binds to DNA and allows to visualize DNA under UV ray. 3. The agarose gel was poured on the casting tray with the comb in place. Allow the gel to set at 40 c for 10-15 minutes or leave at room temperature to set for 20-30 minutes. In this case the gel was kept at 40 c for setting. 4. The combs were removed. 5. The buffer tanks were filled with 1 x TAE buffer till the gel was covered. 6. The DNA extract along with the tagging dye were mixed outside in separate sheets and charged into the wells. 7. The electrophoresis tank was covered and electrophoresis run was done at 80-150v till the tagging dye covered 75-80% of the gel length. 8. The samples were compared with ladder sequence under UV transilluminator.
- 17. 17 Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 17 UV transilluminator PCR can be done on this genomic DNA extract
- 18. 18 Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 18
- 19. 19 Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 19 Other methods Method Best For Pros Cons Phenol-Chloroform General DNA High yield & purity Toxic solvents CTAB Plants Removes polysaccharides Complex Spin Column Clinical/diagnostic Quick & easy Expensive Magnetic Bead Automation Scalable, clean Costly setup Salting-Out General DNA Non-toxic Lower purity Alkaline Lysis Plasmids High plasmid yield Not for genomic DNA
- 20. 20 Use or disclosure of data contained on this sheet is subject to the restriction on the title page of this proposal or quotation. 20 Acknowledgement: Molecular Lab and R &D Dept KIMS Special thanks to Dr Bandita Panda, Research Scientist, KIMS
Editor's Notes
- #6: Lysis buffer (Tris-HCl+ EDTA+ SDS (0.5-2%) or Triton X-100+ NaCl (50-200mM)). Tris-HCl- maintains pH, SDS is the detergent, EDTA-Chelates divalent metal ions (like Mg²⁺), inhibiting DNases that require these ions for activity. NaCl- Maintains ionic strength and helps in protein precipitation.
- #10: AW1-[Guanidine HCl or GuSCN (0.5–1 M)+ Tris-HCl (10 mM, pH 7.5–8.0)+ Ethanol (20–40%)]. Guanidine HCl or GuSCN-Chaotropic salt; helps keep DNA bound to the silica column and denatures proteins. Tris- Maintains the pH, Ethanol- Helps precipitate contaminants and supports DNA binding to column.
- #11: AW2- Ethanol (70–80%)+ Tris-HCl or TE Buffer (optional, low concentration). Ethanol washes away salts and residual proteins without eluting DNA. TE buffer- Maintains pH, but usually minimal to avoid early DNA elution.
- #13: ANY ONE OF THESE. TE Buffer (10 mM Tris, 1 mM EDTA, pH 8.0): Provides long-term DNA stability but may interfere with some enzymatic reactions (due to EDTA).10 mM Tris-HCl (pH 8.0): Preferred when downstream applications like PCR, sequencing, or cloning are planned. Nuclease-free water: Maximizes compatibility, but DNA is less stable long-term (store at −20°C if using water).