Ways-and-Tips-for-DNA-Extraction-and-Purification

1. Ways and Tips for DNA Extraction and Purification CD 5 Ways to Extract and Purify A DNA Sample Phenol-chloroform extraction Phenol-chloroform extraction, also known as organic extraction, uses sodium dodecyl sulfate (SDS) and proteinase K to enzymatically digest proteins and non-nucleic acid cellular components. Then, a mixture of phenol: chloroform: isoamyl alcohol (25:24:1) is added to promote the partitioning of lipids and cellular debris into the organic phase while leaving isolated DNA in the aqueous phase. After centrifugation, the purified DNA in the aqueous phase can be transferred to a clean tube for downstream analysis. Alternatively, DNA can be recovered and concentrated from the aqueous phase via ethanol precipitation or the application of a centrifugal filter unit, so that the DNA in the samples can be further purified and concentrated. Pros: A cheap, reliable and effective way to remove proteins from DNA solutions. DNA Protein Cons: Time-consuming, usage of hazardous chemicals, increased opportunities for contamination and sample mishandling, possibility of inhibiting downstream enzymatic reactions due to phenol/chloroform carry-over. Lipids Cell Lysate Cells DNA Add SDS and Proteinase K Add PCIA Transfer and Retain Aqueous Phase DNA Incubate Vortex and Centrifuge Tel: 1-631-624-4882 Email: info@cdparticles.org

2. Ethanol precipitation Ethanol precipitation is a common way for desalting and concentrating DNA. Both monovalent cations (0.1-0.5 M, usually in the form of the acetate salt of sodium) and ethanol are added to the DNA to a final concentration of 70%. Ethanol could change the DNA structure, leading to the aggregation of the DNA molecules and precipitation out of solution. Most salts and small organic molecules are soluble in 70% ethanol and the precipitated DNA is left to be ready for separation by centrifugation. Pros: A cheap and effective way to desalt and concentrate DNA. Cons: Time consuming, risks of carrying ethanol over into the final sample. Silica column-based kits Loaded lysate Pros: Convenient, relatively fast, the ability to process a large number of samples. Centrifuge Centrifuge Silica Cons: Relatively costly, low yields (as low as 25%), chaotropic salt carry-over. membrane Sample Lysis Column Loading Wash and Dry the Membrane DNA Elution It's a convenient approach to DNA cleanup. The principle is that chaotropic salts are added to the sample to denature the DNA via disrupting its hydrogen bonds. Under this condition, the DNA will selectively bind to the silica resin in the column and thus promoting its separation from the rest of the sample. After washing, a low salt solution is used to elute the DNA from the column, which makes the renaturing of the DNA, leading to its decreasing affinity for the silica. Based on this technology, there are many commercial kits which are ideal for DNA cleanup after applications such as agarose gel extraction, enzymatic reactions and PCR. Tel: 1-631-624-4882 Email: info@cdparticles.org

3. Anion exchange The basic principle is that the positively charged DEAE functionalized resins are able to bind the negatively charged DNA phosphate backbone. By using specific salt in specific pH condition, DNA in the sample will bind the resin, followed by stringent washing steps to remove contaminants such as protein and cellular debris. After the above steps, DNA can be selectively eluted from the resin. Cell Extract Salt More Salt Ion-exchange Resin Pros: High purity DNA, ideal for downstream applications e.g. transfection and DNA sequencing. Protein and RNA Cons: Resins are expensive. Discard DNA Magnetic beads By using magnetic beads, DNA can conditionally bind to them in a pH-dependent manner, which enables you to isolate the DNA from the rest of the sample by simply controlling pH. To be more specific, the magnetic beads are positively charged and bind DNA at low pH, while at high pH they become negatively charged and thus release the DNA. There are a few commercial magnetic bead kits available now. Add Sample Lysis Remove the Supernatant Magnetic Beads Direct Use DNA Elution Pros: Fast, no chaotrophic salts or organic washing solutions required, ideal for automation of high throughput processing. Wash Cons: The initial cost in purchasing the magnets is reasonably high. This procedure can be tricky when handling multiple samples without an automated workflow. Tel: 1-631-624-4882 Email: info@cdparticles.org

4. Tips for DNA Extraction and Purification with Magnetic Beads As one of DNA cleanup options, magnetic beads are simple and effective to fulfill molecular biologists' desire of DNA purification. However, even a simple technique can go wrong and produce sub-optimal DNA end products. To help ensure that the simple stays effective, we introduce you some tips for DNA extraction and purification with magnetic beads. Never freeze your magnetic beads Unless otherwise mentioned, magnetic beads can never ever be frozen. Beads must always be stored at 2 to 8 ℃. There may be cracks on the surfaces of your beads after freezing and thawing, leading to sample contamination. Bring your beads to room temperature Before using your magnetic beads, you should bring them to room temperature. In other words, you need to plan ahead and leave enough time (usually 30 minutes are ideal) for beads to warm up before you need them. 77°F 25℃ 68°F 20℃ Homogenize your magnetic beads Magnetic beads are huge compared to the stuff in molecular biology. Therefore, they may settle into the bottom during storage. However, you need a homogenous bead slurry to conduct experiments, so you should vortex your beads immediately before using them. You may not continue your experiments until the slurry is an even color and all your beads are in suspension, or you may risk endangering your protocol. Pay attention to DNA-bead ratio Bead input is directly correlated to the amount of DNA that you should obtain. Your sample might get contaminative with too many beads, or you might lose product with too few beads. So, you must pay attention to DNA-bead ratio based on the protocol and do not modify randomly. Tel: 1-631-624-4882 Email: info@cdparticles.org

5. Choose an appropriate magnetic stand Whether you are using strip tubes, a 96-well plate, or a MIDI plate, choosing appropriate magnetic stand matters. In addition to the size, stands vary in their magnet placement. Magnet placement affects the aggregation place of your beads, such as the bottom of the well, to the left, to the right, or in a ring conformation. If your protocol doesn't point out which stand is best, you can choose based on your personal preferences. Moreover, you'd better find a stand with good magnetic strength. Pipette carefully Keep your fingers steady and go slow, don’t aspirate too quickly. Monitor your loss Keep an eye on your sample loss at each wash step, so that you may clarify why some samples might work better than others. Creative Diagnostics provides a variety of DNA extraction and purification products, including silica coated magnetic beads , ion-exchange magnetic particles and commercial purification kits. Please visit our website to explore more. For more information, view our website: www.cd-bioparticles.com Email: info@cdparticles.org Address: 451 Tel: 1-631-624-4882 Fax: 1-631-938-8221 Ramsey Road, Shirley, NY 11967, USA 5