CHAPTER 8

Examining DNA from plugs using pulsed-field gel electrophoresis allows one to make sure that the DNA in the plugs is not degraded. If the DNA is not of sufficient length, new plugs will need to be made (possibly using a different nuclear isolation protocol or a different extraction buffer).

Based on relative staining intensity of the samples compared to lambda standards, a mean DNA amount per plug can be estimated.

SUPPLIES, EQUIPMENT, AND REAGENTS (see CHAPTER 2 for details): agarose plugs containing genomic DNA (see CHAPTER 7); 0.5X TBE; agarose; PFGE Lambda Ladder; 10X uncut lambda DNA; blue juice; ethidium bromide; 70% ethanol (in a spray bottle); coverglasses and/or razorblades; CHEF gel apparatus; regular CHEF gel casting apparatus (14 x 13 cm); 15-tooth gel comb; UV light box equipped with camera or image-capture system

METHODS:

1. Make up a 1.0 % agarose/0.5X TBE gel using the regular CHEF gel casting stand and the 15-tooth gel comb. Fill the electrophoresis chamber of the CHEF system with 2.5 L of 0.5X TBE. Set the cooling module so that the buffer is cooled to 12ºC (see CHAPTER 6 for details regarding preparation of gels and operation of the CHEF gel system).
2. Place a slice of the PFGE Lambda Ladder into lanes 3 and 9 of the gel.
3. Place one plug containing the sample DNA onto a clean coverglass (i.e., a coverglass washed with 70% ethanol and dried with a Kimwipe). Using another clean coverglass, cut the plug in half. Take one of the halves and place it into the fifth well of the agarose gel leaving an empty well between the sample and the ladder. Cut the remaining half plug into two pieces (each piece is ¹/₄ of a plug). Take one of these ¼ plug pieces, and place it in the sixth well of the gel. Take the second ¼ plug piece and divide it into two equal pieces. Take one of the resulting ¹/₈ plug pieces, and transfer it into the seventh well of the agarose gel. Discard the remaining ¹/₈ plug.
4. Place 20 µl (10 µg), 10 µl (5 µg), and 5 µl (2.5 µg) of 10X uncut lambda DNA into separate 0.65 ml microcentrifuge tubes. Add MBG water to the second and third tubes so that all tubes contain a total volume of 20 µl. Add 5 µl of blue juice to each tube, mix gently, and spin tubes in a microcentrifuge at 10,000 rpm for 15 sec. Place the complete contents of the tubes containing 10 µg, 5 µg, and 2.5 µg of lambda DNA in lanes 11, 12, and 13, respectively.
5. Run the gel using the following parameters: initial switch time = 1.0 sec, final switch time = 40.0 sec, run time = 18 hours, volts/cm = 6.0, included angle = 120°, ramping = linear.
6. Stain the gel and either photograph or digitally capture its UV fluorescent image. A photograph showing the appearance of a typical gel is presented in FIGURE 8.1. If a photograph is taken, a visual examination of the ethidium bromide fluorescence in the lambda standard lanes compared with the fluorescence in the sample lanes can be used to estimate the DNA content of a plug. If the image has been digitally captured, imaging software can be used to determine the amount of DNA in a typical plug.

• If the mean DNA length is > 600 kb, the plugs should be further evaluated (CHAPTER 9).
• If the mean DNA length of the test DNA is < 600 kb, it is likely that the DNA in the plugs has degraded too much to be of use in BAC library construction. Consequently, new plugs should be prepared.

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