Isolating Genomic DNA using PureLink Kits

DNA Extraction and Purification

• The PureLink Genomic DNA Kits allow for rapid and efficient purification of genomic DNA from various sources, including blood samples.
• The kit is designed to isolate genomic DNA from mammalian cells and tissues, such as mouse/rat tail, blood samples, buccal swabs, bacteria, and yeast.
• The extracted DNA is suitable for various downstream applications, including PCR, restriction enzyme digestion, and Southern blotting.
• The isolated DNA is 20–50 kb in size.

Preparing Lysates

• To prepare a lysate, a fresh or frozen blood sample is added to a sterile microcentrifuge tube.
• Proteinase K and RNase A are then added to the sample, followed by incubation at room temperature for 2 minutes.
• The sample is then incubated at 55°C for 10 minutes to promote protein digestion.
• Ethanol is added to the lysate, and the mixture is vortexed for 5 seconds to yield a homogenous solution.

Binding DNA

• The lysate is mixed with PureLink Genomic Lysis/Binding Buffer and ethanol, and the mixture is vortexed for 5 seconds.
• The resulting solution is then bound to a silica-based membrane in a column or plate.
• Impurities are removed by thorough washing with Wash Buffers.
• The genomic DNA is then eluted in low salt Elution Buffer.

DNA Restriction (Digestion)

• Restriction enzymes are used to specifically cut long pieces of genomic DNA into manageable fragments.
• Each restriction enzyme has its own specific recognition site on double-stranded DNA, usually 6 to 8 bp in length and palindromic in sequence.
• Typical digestions include a unit of enzyme per microgram of starting DNA, and one enzyme unit usually is defined as the amount of enzyme needed to completely digest one microgram of double-stranded DNA in one hour at the appropriate temperature.

Gel Electrophoresis

• Gel electrophoresis is a powerful technique in molecular biology that separates molecules based on their charge, size, and other physical features.
• DNA molecules are negatively charged on their phosphate groups and travel from the negative pole to the positive pole in an electric field.
• The gel functions as a sieve through which DNA migrates to a distance inversely proportional to its molecular weight.
• The resulting bands can be visualized using ethidium bromide staining, which intercalates in the DNA double helix and gives a fluorescent glow upon exposure to UV.

Laboratory Techniques and Precautions

• Various laboratory techniques are used in molecular biology, including PCR, restriction enzyme digestion, and Southern blotting.
• General techniques and precautions include solution preparation, DNA extraction and electrophoresis, DNA digestion and electrophoresis, competent cell preparation, bacterial transformation, molecular cloning, and screening for recombinants.
• Personal protective equipment, such as gloves and eye protectors, should be worn when handling mutagenic and carcinogenic materials like ethidium bromide and UV.