Separating+DNA+Fragments+Very+Close+in+Size

Separating DNA Fragments Very Close in Size

Excerpt from http://delliss.people.cofc.edu/virtuallabbook/LabReadings/Miniprep/MINIPREPARATION.pdf 2. Fragment Resolution There are limits to the ability of agarose to separate DNA fragments of different sizes. When two fragments are close together in size, they may appear as one band in the gel rather than two. For example; if you expect 6 DNA fragments and thus 6 bands on a gel but you see only 5 bands, then it is likely that two of the fragments have not resolved in the gel. It is termed a doublet when two fragments appear as one band. Carefully examine Figure 5.1 and notice that there are two doublet bands.

Agarose gels separate DNA fragments differing by a hundred or more base pairs, while polyacrylamide gels can separate DNA fragments differing by a single base pair. excerpt from http://biology.arizona.edu/sciconn/lessons2/Vuturo/vuturo/gel.htm

Excerpts from http://www.msptm.org/files/351_-_354_Lee_S_V.pdf > agarose provide better resolution for large > fragments by providing greater separation > between bands that are close in size. High > gel concentrations, on the other hand, > reduce the migration speed of the long > fragments while facilitating better > separation of small DNA fragments. > the best resolution of fragments larger than > 2 kb is attained by applying no more than 5 > V/cm to the gel. > Combination of high and low > agarose concentrations would facilitate > distinct migration of both smaller and > larger fragments of DNA at a consistent voltage (4.5 V/cm).
 * Low concentrations of
 * Note they developed some new method

Pulsed Field Electrophoresis Unlimited increase in the resolution of DNA ladders.
 * http://en.wikipedia.org/wiki/Pulsed_field_gel_electrophoresis
 * The procedure for this technique is relatively similar to performing a standard gel electrophoresis except that instead of constantly running the voltage in one direction, the voltage is periodically switched among three directions; one that runs through the central axis of the gel and two that run at an angle of 120 degrees either side.
 * These two stages of electrophoresis are serially repeated. Eventually, both the short and the long DNA ends of the ladder migrate out of the gel while a selected region of the ladder undergoes progressive increase in resolution during back-and-forth migration. Improved resolution of DNA bands is achieved, without a known limit.
 * electrophoresis buffer was equilibrated against the gel for at least 2 h

http://www.peds.ufl.edu/divisions/cellular/zolotukhin/PDF/cpmb_2.5B.pdf > pump. > showed that it was possible to resolve molecules > that would otherwise run at limiting mobility > by cyclically varying the orientation of > the electric field in the gel during the run. > separates only a relatively narrow range of > sizes. To get resolution over a broad size range, > it is necessary to use a range of reverse times; > this is accomplished with a time ramp—i.e., > progressively increasing the forward and reverse > intervals from a lower limit to an upper > limit. > length. > ~80% of the power-supply voltage to the > gel (the rest is lost in the buffer tanks), so > voltage can be estimated by multiplying the > power supply readout by 0.8 and dividing the > result by the length of the gel. > V/cm > used, it is critical that the entire gel be at the > same temperature to prevent the “smile” effect. > This is best accomplished by recirculating the > buffer using a peristaltic pump. In many cases, > pulsed-field gels can be run on the benchtop > with no cooling. Gels that generate a lot of heat > can often be run in a cold room. > TAE carries a lot of current, causing gels to heat up. > of molecules, possibly by making > DNA stiffer. Addition of ethidium bromide to > the gel can help resolve molecules <100 kb, but > is not recommended for larger molecules. > the sizes of molecules resolved toward the > larger range, and also speeds the migration of > all molecules. > generally in the range of 2.5:1 to 3.5:1 > longer time, at lower voltage, or both. > than can be dissipated effectively. Either > reduce the heating by decreasing voltage, gel > thickness, or buffer depth, or increase buffer > recirculation.
 * Current Protocols Protocol Link**
 * The most commonly used ratio of forward to reverse time is 3:1
 * Allow bromphenol blue to migrate 1 cm, then start switching device and peristaltic
 * Schwartz and Cantor (1984)
 * in field inversion a single pulse duration
 * Voltage is measured in V/cm of gel
 * Most horizontal submarine gel boxes apply
 * Pulsed-field gels are generally run at 5 to 10
 * Note: Cause of "smiling effect": Although different temperatures may be
 * DNA has higher mobility in TAE buffer, but
 * Ethidium bromide. Ethidium slows the reorientation
 * Agarose. Low agarose concentration shifts
 * best to use a time ramp that is just long enough
 * The ratio between forward and reverse times is
 * Bands smeared. Run samples into gel for
 * Excessive smile. More heat is being produced