CHAPTER 14

During ligation, insert DNA is ligated with vector DNA to form BACs. A test transformation followed by a miniprep/NotI analysis provides information on the success of ligation and transformation using plant DNA as the source of inserts.

SUPPLIES, EQUIPMENT, AND REAGENTS (see CHAPTER 2 for details): dephosphorylated pBeloBAC11 vector stock (see CHAPTER 3); insert DNA (see CHAPTER 13); T4 ligase; 10X T4 ligase buffer; 10% PEG; Millipore nitrocellulose filters; 70% ethanol; ElectroMax® DH10B^TM competent cells; SOC; X/I/C Petri plates; glass plating rod; electroporator with cuvettes; MP-1; MP-2; MP-3; LB^+CM; isopropanol (stored at –20ºC); 70% ethanol (-20ºC); 0.5X TBE; PFGE Midrange Ladders or PFGE Lambda Ladder; agarose; NotI with 10X buffer and 100X BSA; blue juice; sterile toothpicks; CHEF gel apparatus; large CHEF gel casting stand; 45-tooth gel comb; UV light box equipped with camera or image-capture system

METHODS:

1. Place insert DNA, the dephosphorylated pBeloBAC11 vector stock solution, and the T4 ligase 10X buffer on ice. Allow the buffer and the vector stock to thaw. Keep the T4 ligase in the –20ºC freezer until immediately before use.
2. Set up ligation reactions as described below. The number of reactions that can be prepared depends upon the nanograms of insert DNA available. In general, we make up 150 µl reactions in 1.5 ml microcentrifuge tubes as follows:

Ligation reaction

50 ng vector DNA

15 µl 10X T4 ligase buffer

3 µl T4 ligase (i.e., 9 units)

300 ng of insert DNA

MBG water to give a final reaction volume of 150 µl

­ Note 14.1: Most BAC libraries have been constructed using a molar ratio of 5-15 parts size-selected DNA to 1 part BAC vector. We typically start off using a 5:1 ratio. If a 5:1 ratio does not produce a satisfactory outcome, changing the ratio of insert to vector can sometimes improve the results.

3. Gently tap each reaction tube to mix the tube's contents. DO NOT VORTEX OR AGITATE VIOLENTLY AS THIS MAY SHEAR THE INSERT DNA.
4. Incubate the ligation reactions at 16ºC overnight as described in CHAPTER 4.
5. Place ligation reaction tubes in a 65ºC water bath for 20-30 min to "heat kill" the enzyme.
6. Desalt the ligated DNA as described in CHAPTER 4. Place no more than 300 µl of ligation reaction on any particular Millipore nitrocellulose filter.
7. Using a pipettor and large-orifice pipet tips, transfer all of the desalted ligation reactions into a single 1.5 ml microcentrifuge tube (i.e., pool the ligation reactions).

­ Note 14.2: Due to osmosis during desalting, the total volume of liquid placed on each filter typically will be one-third to one-half that of the starting volume.

8. Place the tube at 4ºC.

­ Note 14.3: Ligated DNA is stable at 4°C for at least 5 days.

9. Perform a test transformation exactly as described in CHAPTER 5 except expose the contents of each cuvette to 320-330 volts rather than 390-400 volts.
10. After incubating test plates overnight, determine the titer of the transformation reaction and the percentages of white and blue colonies. If more than 60% of the colonies are white, select thirty-six white colonies from the test plates and perform a NotI digestion on isolated BACs exactly as described in CHAPTER 6. A diagram illustrating the appearance of a successful transformation/miniprep/NotI digest is shown in FIGURE 6.1. Photographs of BAC NotI digests from three plant species are shown in FIGURE 14.1.

    ­ Note 14.4: Dicots contain few NotI restriction sites in their DNA. Thus, NotI digests of dicot BACs produce only one or two bands (in addition to the vector band) on agarose gels. In contrast to dicots, NotI sites are more frequent in monocot DNA, and consequently more bands are observed in BAC NotI digests from monocots (see FIGURE 14.1). Because of the often numerous bands in NotI digests of monocot inserts, it is best to use the PFGE Midrange Ladders as size standards rather than the PFGE Lambda Ladder. The PFGE Midrange Ladders possess more fragments covering a wider range of fragment sizes than the PFGE Lambda Ladder, and thus they allow more accurate determinations of insert size in species containing numerous NotI sites.

• If the percentage of false positive clones is > 10% it is possible that the vector DNA was damaged during dephosphorylation. An extremely high percentage of blue clones (> 40%) suggests possible problems during vector preparation, ligation, and/or transformation.
• The combined base pair lengths of all non-vector bands in a particular lane constitute the length of the insert (see FIGURE 6.1 and FIGURE 14.1). What is the mean insert length? What is the size of the largest insert? What is the size of the smallest insert? Whether clones contain inserts of adequate size is based upon the needs of those who intend to use the library. The protocol presented in this paper is designed to generate clones with insert sizes between 100 kb and 350 kb. However, smaller insert sizes may be sufficient (or even preferable) for certain applications.

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