Performance Typical DNA Recoveries from Agarose Gels DNA Size (bp) 100 700 1000 2027 4361

9416 23130 Percent DNA Recoveries 78 71 77 47 35 32 29

Digestion with Multiple Enzymes Digesting DNA with two enzymes is a commonplace task, and oftentimes the two enzymes have different buffer requirements. There are at least three ways to handle this situation:

Digest with both enzymes in the same buffer. In many cases, even those a given buffer is not optimal for an enzyme, you can still get quite good cleavage rates. Enzyme manufacturer catalogs usually contain a reference table recommended the best single buffer for conducting specific double digests. Cut with one enzyme, then alter the buffer composition and cut with the second enzyme. This usually applies to situations where one enzyme like a low salt buffer and the other a high salt buffer, in which case you can digest with the first enzyme for a time, add a calculated amount of concentrated NaCl and cut with the second enzyme. Change buffers between digestion with two enzymes. In some cases, two enzymes will have totally incompatible buffers. In that case, perform one digestion, recover the DNA (usually by precipitation) and resuspend in the buffer appropriate for the second enzyme

Carriers Carriers (or coprecipitants) are inert substances that are used to improve the recovery of small quantities of nucleic acids during ethanol precipitation. Insoluble in ethanolic solutions, carriers form a precipitate that traps the target nucleic acids. During centrifugation, carriers generate a visible pellet that facilitates handling of the target nucleic acids. This may be their major virtue: As discussed above, ethanol precipitation - even of small amounts of nucleic acids in dilute solution - is remarkably efficient. Carriers do little, other than provide visual clues to the location of the target nucleic acid. Three substances are commonly used as carriers: yeast tRNA, glycogen, and linear polyacrylamide. Their advantages and disadvantages are listed in Table A8-2. Glycogen is usually used as a carrier when nucleic acids are precipitated with 0.5 M ammonium acetate and isopropanol. Glycogen is not a nucleic acid and therefore does not compete with the target nucleic acids in subsequent enzymatic reactions. However, it can interfere with interactions between DNA and proteins (Gaillard and Strauss 1990).

Ethanol precipitation is a commonly used technique for concentrating and de-salting nucleic acid (DNA or RNA) preparations in aqueous solution. The basic procedure is that salt and ethanol are added to the aqueous solution, which forces the nucleic acid to precipitate out of solution. The precipitated nucleic acid can then be separated from the rest of the solution by centrifugation. The pellet is washed in cold 70% ethanol then after a further centrifugation step the ethanol is removed, and the nucleic acid pellet is allowed to dry before being resuspended in clean aqueous buffer. So how does this work?

A bit about solubility First we need to know why nucleic acids are soluble in water. Water is a polar molecule it has a partial negative charge near the oxygen atom due the unshared pairs of electrons, and partial positive charges near the hydrogen atoms (see the diagram on the right). Because of these charges, polar molecules, like DNA or RNA, can interact electrostatically with the water molecules, allowing them to easily dissolve in water. Polar molecules can therefore be described as hydrophilic and non-polar molecules, which cant easily interact with water molecules, are hydrophobic. Nucleic acids are hydrophilic due to the negatively charged phosphate (PO3-) groups along the sugar phosphate backbone. The role of the salt

Ok, so back to the protocol. The role of the salt in the protocol is to neutralize the charges on the sugar phosphate backbone. A commonly used salt is sodium acetate. In solution, sodium acetate breaks up into Na+ and [CH3COO]-. The positively charged sodium ions neutralize the negative charge on the PO3- groups on the nucleic acids, making the molecule far less hydrophilic, and therefore much less soluble in water. The role of the ethanol The electrostatic attraction between the Na+ ions in solution and the PO3- ions are dictated byCoulombs Law, which is affected by the dielectric constant of the solution. Water has a high dielectric constant, which makes it fairly difficult for the Na+ and PO3- to come together. Ethanol on the other hand has a much lower dielectric constant, making it much easier for Na+ to interact with the PO3-, shield its charge and make the nucleic acid less hydrophilic, causing it to drop out of solution. The role of temperature Incubation of the nucleic acid/salt/ethanol mixture at low temperatures (e.g. -20 or -80C) is commonly cited in protocols as necessary in protocols. However, according to Maniatis et al (Molecular Cloning, A Laboratory Manual 2nd Edition 2nd edition?? I need to get a newer version!), this is not required, as nucleic acids at concentrations as low as 20ng/mL will precipitate at 0-4C so incubation for 15-30 minutes on ice is sufficient. The wash step with 70% ethanol This step is to wash any residual salt away from the pelleted DNA. A few tips on nucleic acid precipitation

Choice of salt Use Sodium acetate (0.3M final conc, pH 5.2) for routine DNA precipitations

The pET System is the most powerful system yet developed for the cloning and expression of recombinant proteins in E. coli. Target genes are cloned in pET plasmids under control of strong bacteriophage T7 transcription and (optionally) translation signals; expression is induced by providing a source of T7 RNA polymerase in the host cell. T7 RNA polymerase is so selective and active that, when fully induced, almost all of the cells resources are converted to target gene expression; the desired product can comprise more than 50% of the total cell protein a few hours after induction. Although this system is extremely powerful, it is also possible to attenuate expression levels simply by lowering the concentration of inducer. Decreasing the expression level may enhance the soluble yield of some target proteins. Another important benefit of this system is its ability to maintain target genes transcriptionally silent in the uninduced state. Target genes are initially cloned using hosts that do not contain the T7 RNA polymerase gene, thus eliminating plasmid instability due to the production of proteins potentially toxic to the host cell (see Section I. F. for details). Once established in a non-expression host, target protein expression may be initiated either by infecting the host with CE6, a phage that carries the T7 RNA polymerase gene under the control of the pL and pI promoters, or by transferring the plasmid into an expression host containing a chromosomal copy of the T7 RNA polymerase gene under lacUV5 control. In the second case, expression is induced by the addition of IPTG to the bacterial culture. Although in some cases (e.g., with innocuous target proteins) it may be possible to clone directly into expression hosts, this approach is not recommended as a general strategy. Two types of T7 promoter and several hosts that differ in their stringency of suppressing basal expression levels are available, providing great flexibility and the ability to optimize the expression of a wide variety of target genes.

The Ultrafree-DA device is designed to recover 100 to 10,000 bp DNA from agarose gel slices in one 10-minute spin. It consists of a pre-assembled sample filter cup with agarose Gel Nebulizer, and a microcentrifuge vial. The device utilizes gel compression to extract DNA from the agarose. Centrifugal force collapses the gel structure, drives the agarose through a small orifice in the Gel Nebulizer and the resultant gel slurry is sprayed into the sample filter cup. As the agarose is compressed at 5,000xg, DNA is extruded from the gel's pores. The gel matrix is retained by the microporous membrane, and the DNA passes freely through the membrane. DNA can then be recovered in the filtrate vial.