DNA Isolation Basic Theory

DNA ISOLATION Basic theory Many different methods and technologies are available for the isolation of genomic DNA. In general, all methods involve destruction and lysis of the sample followed by removal of proteins and other contaminants to produce pure DNA. The protein is removed with the proteinase K enzyme, followed by salt-out, organic extraction, or binding of the DNA to the solid phase (either anion exchange or silica technology). DNA is precipitated using ethanol or isopropanol The choice of method depends on many factors: the required DNA quantity and molecular weight, the desired DNA purity for subsequent application, time and cost. Separation of DNA from cellular components can be divided into four stages: 1. Sample preparation to collect cell harvest 2. Cell Lysis 3. Digestion of proteins so that nucleic acids are released (protein digestion) 4. DNA precipitation (DNA preparation) Silica-based methods Qhe Genomic DNA Mini Kit (Blood/Cultured Cell) provides an efficient method for purifying total DNA (including genomic, mitochondrial and viral DNA) from fresh whole blood, cultured animal cells,buffy coat, yeast and others. Chaotropic salts are used to lyse cells and digest proteins, allowing DNA to bind to the glass fiber matrix of spin (1). Contaminants are removed using Wash Buffer containing ethanol. Purification of genomic DNA by eluting with low salt Elution Buffer. The whole procedure can be completed within 25 minutes without phenol/chloroform extraction or alcohol precipitation. The purified DNA, with approximately 20-30 kb, is suitable for use in PCR or other enzymatic reactions (Figure 1). The purity of the isolated DNA is analyzed quantitatively using a spectrophotometer and checked by electrophoresis. From the entire 200 mlFresh human blood can produce 4-6 mg pure DNA with an A260/A280 ratio of 1.6-1.8. Salting out method It begins with lysis of the cell membrane. Salting out is a conventional technique where proteins and contaminants are precipitated through lysis of cell membranes using high concentration salts such as potassium acetate or ammonium acetate. The precipitate was removed by centrifugation, and alcohol precipitation was carried out to reveal DNA. The use of this method for the removal of impurity compounds such as protein and RNA is considered likely to be less efficient. The purity of the DNA obtained by this method varies greatly. Molecular biology analysis techniques are one of the keys to understanding the central dogma in biology. The existence of this technique will help us understand the processes of replication, transcription, translation, etc. This technique allows us to understand organisms at the molecular level, namely the DNA, RNA, amino acid and protein levels. The technique that will be discussed in this practicum is PCR (Polymerase Chain Reaction).PCR is a technique for synthesizing and duplicating DNA and RNA in vitro. PCR was first developed by Karl Mullis in the mid-1980s. PCR is based on exploiting the ability of DNA polymerase to synthesize new strands of complementary DNA from DNA templates. The mechanism that occurs during PCR is the same as DNA replication in vivo. The PCR process occurs over several cycles. The PCR method can increase the number of DNA sequences thousands or even millions of times from the original number, around 106107time. The components needed in the PCR reaction are DNA template, primers (oligonucleotides), and PCR mix. DNA template is a number of DNA samples that will become a template for the formation of new DNA strands during the amplification process using the PCR technique. A primer is a short sequence of DNA or RNA (oligonucleotide) that is complementary to the template DNA strand used to initiate DNA amplification. PCR mix contains PCR buffer, Mg2+, dNTP, and polymerase enzyme. Tools and materials Material: 1. Blood from a vein that has been given an anticoagulant (EDTA/ Ethylenediaminetetraacetic acid) 2. Oral mucosal epithelial cell solution Tool: 1. Micropipette 2. microcentrifuge tube size 1.5 ml 3. Blue tip, yellow tip and white tip 4. Centrifuge tube 5. Centrifuge 6. Spectrophotometer 7. The incubator is set at 60°C 8. Water bath set to 37°C Reagent 1. Genomic DNA Mini Kit 2. Absolute ethano Ways of working Step I. Sample preparation 1. Take 300 µl of blood sample and put it inmicrocentrifuge tubesize1.5 ml. 2. Add 900 µl of RBC lysis buffer then mix by inversion (back and forth). 3. Incubate for 10 minutes at room temperature. 4. Then centrifuge for 5 minutes at a speed of 3000 g, then discard the supernatant. 5. Add 100µl RBC lysis buffer to the pellet and then resuspend until homogeneous STEP II. Cell Lysis 1. Add 200µl GB buffer insidemicrocentrifuge tubeand shake gently. 2. Incubate in an incubator at 60C for 10 minutes, to ensure the pellets are completely clean. During incubation, shake gently every 3 minutes.(At this stage, incubate the Elution Buffer (100µl per sample) at a temperature of 60 C.STEP III. DNA BINDING 1. Add 200 µl absolute ethanol to the pellet then shake gently for 10 secondsNote: If precipitates appear, remove them with a pipette 2. Place the GD Column in a 2 ml Collection Tube 3. Transfer all the mixture (including precipitates) into the GD Column. 4. Centrifuge at a speed of 10,000 g for 5 minutes, then discard the supernatant in thecollection tube.Washcollection tubewith absolute ethanol then place the GD Column on top. STEP IV WASH 1. Add 400 µl W1 Buffer into the GD Column then centrifuge10,000 g for 1 minute. 2. Discard the supernatant then washcollection tubewith absolute ethanol and place the GD Column on top 3. Add 600 µl Wash Buffer (make sure ethanol has been added) into the GD Column, then centrifuge at 10,000 g for 1 minute then discard the supernatant then place the GD Column on top again. 4. Centrifuge again for 3 minutes at 10,000 g to dry column matrix STEP V. DNA Elution 1. Move the dry GD Column to the topcollection tubewhich is clean, then add 100 µl of Elution Buffer which has been incubated right in the middle column matrix. 2. Leave it for 3 minutes to ensure the Elution Buffer is completely absorbed 3. Centrifuge at 10,000 g for 30 seconds to elute the purified DNA. Testing the Purity of Isolated DNA Measure the absorbance of the supernatant at wavelengths of 260 nm and 280 nm. Write down the absorbance results to calculate the purity of the DNA.