CHAPTER 13

High molecular weight restriction fragments must be removed from surrounding agarose before they can be used in ligation reactions. Presented below are two methods for isolating DNA from agarose plugs.

At present, there are several methods for isolating high molecular weight restriction fragments from agarose. In our experience, both electroelution and GELase digestion work well. Thus each of these methods will be discussed in this chapter. Once size-selected DNA is isolated from LMP agarose blocks it can be ligated into an appropriate vector and used in transformation.

SUPPLIES, EQUIPMENT, AND REAGENTS (see CHAPTER 2 for details): agarose cubes q, r, and s containing size-selected DNA (see CHAPTER 12); electroelution system

METHODS:

In electroelution, a voltage is applied to a gel slice containing DNA. As in standard electrophoresis, the DNA in the agarose moves towards the positive electrode. The DNA exits the gel slice and is collected in a filter cup. DNA solutions removed from filter cups are ready for use in POST-ISOLATION PROCEDURES (see II below).

There are numerous electroelution devices available. Because these devices differ with regard to design and operation instructions, consult the manufacturer's instructions that come with your electroelution apparatus to get optimal results. The BioRad Electroelution system is commonly used in molecular biology research, and consequently detailed instructions for using this system are given in APPENDIX B.

­ Note 13.1: Do not use an electroeluter that previously has been used to isolate high-copy plasmid DNA when trying to isolate high molecular weight DNA. This can lead to sample contamination and confusing results.

Electroelution also can be performed using dialysis tubing and a standard horizontal gel apparatus (see Strong et al. 1997 for details).

SUPPLIES, EQUIPMENT, AND REAGENTS (see CHAPTER 2 for details): agarose cubes q, r, and s containing size-selected DNA (see CHAPTER 12); GELase^TM (0.2 units/µl) with 50X buffer

METHODS:

1. Place a clean weighing boat on a balance. Tare the balance and place one of the three q agarose cubes onto the boat. Record the weight of the agarose cube. Transfer the cube to a sterile 15 ml culture tube.
2. Repeat step 1 using an r cube and an s cube.
3. Add MBG water to each tube. Place the tubes on ice. After 10 min, decant the water and add fresh MBG water. Repeat this process five times. This step removes electrophoresis buffer from the cubes (which possibly could interfere with ligation).
4. Decant the final wash. For every 50 mg of agarose in a particular tube, add 1 µl of 50X GELase buffer.
5. Place the culture tubes in a 70ºC water bath for 3 min (or until all of the agarose has become liquid). Quickly transfer the tubes to a 45ºC water bath
6. Add GELase to each tube so that there is 1.0 unit of enzyme for every 200 mg of agarose.
7. Gently swirl the contents of each tube. Incubate the tubes at 45ºC for 45 min. The DNA in each tube is now ready for use in POST-ISOLATION PROCEDURES (see II below).

1. POST-ISOLATION PROCEDURES

SUPPLIES, EQUIPMENT, AND REAGENTS (see CHAPTER 2 for details): DNA isolated from q, r, and s agarose cubes (see above); 1X TAE; agarose; 70% ethanol in a spray bottle, 1X uncut lambda DNA; submarine mini-gel apparatus; UV light box equipped with camera or image-capture system; blue juice; ethidium bromide

METHODS:

1. Place q, r, and s DNA samples isolated using electroelution or GELase digestion ("insert DNA solutions") in their own 1.5 ml microcentrifuge tubes on ice.
2. Prepare a 1% agarose submarine mini-gel in 1X TAE. In preparing the gel, use a comb with at least 7 teeth.
3. Place 0.5, 1.0, 2.0 µl, and 4.0 µl of 1X uncut lambda DNA (i.e., 25 ng, 50 ng, 100 ng, and 200 ng) in separate 0.65 ml microcentrifuge tubes. Add 2.0 µl of blue juice to each.
4. Take a 5 µl aliquot from the tube containing q insert DNA and place the solution in a 0.65 ml microcentrifuge tube. Add 2 µl of blue juice. Do the same for the r and s samples.
5. Submerge the mini-gel in 1X TAE buffer in an appropriate mini-gel apparatus. Load the gel as shown in FIGURE 13.1. Run the gel at 100 v for 15-20 min. Stain and photograph the gel as described in CHAPTER 3. Based on comparison of the relative fluorescence in the sample and standard lanes, an estimate of the concentration of each sample can be made (FIGURE 13.1). Multiplication of a sample's volume by its concentration gives an estimate of the total amount of insert DNA in that sample.
6. Select the insert DNA solution with the greatest concentration of high molecular weight DNA for use in subsequent ligation reactions. In our experience, the q solution generally has a DNA concentration of 10-20 ng/µl while the r and s solutions have concentrations substantially below 10 ng/µl. However, any of the solutions with a DNA concentration > 5.0 ng/µl can be used in ligation.

   ­ Note 13.2: If the insert solutions are particularly dilute (< 5.0 ng/µl), they can be concentrated using Millipore nitrocellulose filters and 10% PEG [see step 6 ("desalting") in CHAPTER 4; also see FIGURE 4.1].

   ­ Note 13.3: High MW (insert) DNA samples are quite unstable. Though they can usually be left at 4°C overnight, it is best to perform ligation immediately after checking the DNA concentration.

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