BIO 141.docx

BIO 141 MODULE 2 Restriction Enzyme Digests Retrieve the restriction enzyme from the fridge. Keep restriction enzyme on ice or thermoresistant container to make sure there is optimal activity for future reactions. Microfuge tubes: a volume of sterile nuclease free water that will yield a final react...

BIO 141 MODULE 2 Restriction Enzyme Digests Retrieve the restriction enzyme from the fridge. Keep restriction enzyme on ice or thermoresistant container to make sure there is optimal activity for future reactions. Microfuge tubes: a volume of sterile nuclease free water that will yield a final reaction volume of 20 microliters. 10x restriction buffer, BSAM 1 microgram of DNA, 2-10 units of enzyme. 1 unit = 1 microgram DNA digested in 1 hr of 37C in 50 microliter. Mix by vortexing, centrifuge briefly at 12000 time g in a microcentrifuge. Incubate at an optimal temperature for restriction enzyme: 37C in a heating block for 1-4 hours. Incubate the reaction mixture at 65 C to heat and activate restriction enzyme. Prolonged incubation times may result to star activity: cutting at sites that is similar but distinct from typical digestion sites. Following inactivation, DNA should be run in an agarose gel to ensure that the digest was successful. Sometimes multiple enzymes have to be used to generate a specific DNA fragment. Check if buffer conditions and incubation temperatures are compatible between two enzymes. If so, double digest may be performed. Use enzymes sequentially if conditions are not compatible. Purify DNA following initial digest, then do a second digest. The use of control DNA with known restriction sites allows the enzyme activity to be tested. Applications: Screening Samples: can be used diagnostically to identify samples. Restriction Fragment Length Polymorphisms (RFLPs) Subcloning – isolate a fragment of DNA in one plasmid and insert it into another so the desired fragment can be replicated using bacteria. Determine the presence of single nucleotide differences in alleles. Agarose Gel Electrophoresis Agarose Gel Electrophoresis is a technique commonly used to separate DNA molecules by their size. Agarose, a polysaccharide derived from red algae is added to a buffer such as tris-acetate EDTA (TAE) or Tris-borate EDTA (TBE) and dissolved by heating. 1 gram/100 mL = 1% agarose solution. To stain the gel, a fluorescent dye such as ethidium bromide is often added to the agarose solution. The solution is poured into horizontal modes. A comb is inserted into the gel cast to create wells to which DNA samples can be loaded. The agarose solution is left undisturbed. The gel consists of hydrogen bonded agarose molecules forming a porous matrix to which the DNA molecules move. DNA samples are mixed with a loading buffer and loaded onto the gel. The loading buffer consists of glycerol, which increases the density of DNA samples and help them settle at the bottom of the well. They also contain dyes, which monitor migrating DNA bands. Negatively charged DNA migrates to the positive electrode. Larger DNA molecules move more slowly than smaller molecules allowing size separation. Size depends on agarose concentration. Higher agarose concentration = smaller pore size. Integration: DNA Isolation and Restriction Enzyme Analysis DNA extraction is the removal and the purification of DNA from dells. First, cells are lysed: broken open by a combination of physical and chemical treatment, the treatment of detergents which dissolves cell and nuclear membrane. STS or sodium dodecyl sulfate is a commonly used detergent that works by solubilizing proteins and lipids that make up these membranes. DNA must be separated from the other molecules present. Proteinase K added to the reaction will break down peptide bonds and digest contaminating proteins. Salt is added which stabilizes the negative charged phosphate groups in the backbone of the DNA, and after the addition of ice-cold alcohol, precipitates the DNA out of the solution. White precipitate is collected by spinning in a centrifuge which settles in the bottom of the tube. Washing and resuspension. DNA Fingerprinting – can identify novel patterns in DNA Subtopic