DNA EXTRACTION METHODS
Extraction of DNA Dry Blood Spot Using Proteinase K Extraction method
1. Cut out bloodstained paper sample
2. Placed in labeled centrifuge tube
3. Cell Lysis- lysis buffer & Proteinase K (release dna)
4. Incubate- digestion of proteins
5. Heated Block- inactivate pK
6. Centrifugation- Separate DNA containing supernatant
7. Precipitation- Ethanol
8. Purification- Ethanol washing
9. Dry DNA pellet
10.Resuspend DNA pellet in TE buffer for prevention
Extraction of DNA from Hair Sample using Phenol-Chloroform DNA Extraction
Method
1. Cell lysis- lysis buffer + Tris-HCl+MgCl2 & Triton X-100
2. Heat- bweaken cell structure
3. Digestion buffer- SDS. DTT & pK
4. Incubated
5. Purification- misture of phenol, chloroform and isoamyl alcohol
6. Centrifuged
7. Supernatant transffered to clean tube
8. Rnase A added- eliminate RNA contamination
9. Precipitation- isopropanol & sodium acetate
10.Incubate
11.Centrifuge
12.Purifictaion- pellet washed w/ ethanol
13.Air dry
14.Resuspended in TE Buffer
Extraction of DNA from a bacterial solution using silica gel-based DNA
extraction method
1. Bacterial culture preparation - centrifuged- supernatant discarded- bacterial pellet
resuspended
2. Cell lysis- lysis buffer & pK
3. Incubated (occasional vortexing)
4. Addition of RNase A & 2nd incubation
5. DNA Binding- binding buffer added enhance DNA interaction w/ silica membrane
6. Precipitation- Ethanol
7. Silica- based spin column
8. Centrifuge
9. Purification- wash buffer 1&2
10.Final Centrifugation
11.DNA Elution- elution buffer+Incubation+final centrifugation
12.Assessed using spectrophotometer - purity & concentration
13.Agarose gel electrophoresis- confirm DNA integrity
Extraction of DNA from urine sample using phenol-chloroform DNA extraction
method
1. Centrifuge urine - discard supernatant & left w/ cell pellet
2. Cell lysis- lysis buffer (SDS) & pK
3. Mixed w/ vortex
4. Incubated
5. Phenol-chloroform extraction- equal volume of phenol:chloroform:isoamyl alcohol
6. Vortexing
7. Centrifuge - 3 layers- the upper aqueous phase (containing DNA), an interphase
(containing proteins), and a lower organic phase (containing lipids)
8. Aqueous phase transferred new tube
9. Precipitation- Sodium Acetate + Isopropanol
10.Incubation
11.Centrifuged- dna pellet
12.Purification- Washed w/ ethanol
13.Vortexed
14.Centrifuged
15.Air-dry pellet
16.Resuspended in TE buffer
Genomic DNA Extraction from FFPE Tissues Using the Proteinase K Method
1. Deparaffinization- tissue section cut using microtome & transffered to
microcentrifuge tube
2. Xylene is added - vortexing-incubation-dissolve paraffin
3. Centrifugation
4. Supernatant is removed
5. Tissue pellet is washed- ethanol
6. Tissue Pellet is air dried
7. Cell Lysis
8. Centrifugation
9. Incubation
10.Precipitation- ethanol
11.Centrifuge
12.Purification Pellet is washed ethanol
13.Air dry
14.Resuspend in nuclease free water