CHAPTER 4

Test ligation

PURPOSE

It is important to see if the vector DNA prepared in CHAPTER 3 is of sufficient quality for use in preparing a BAC library. However, it is generally not a good idea to perform the test ligation with DNA from which you plan to make the library – if the ligation fails, it will not be clear whether the fault is due to the vector or the insert DNA. Rather, the vector can be tested by ligating it with easily obtainable DNA known to be of high quality (e.g., lambda phage restriction fragments).

EXPERIMENTAL PROCEDURES

SUPPLIES, EQUIPMENT, AND REAGENTS (see CHAPTER 2 for details): dephosphorylated pBeloBAC11 vector stock (see CHAPTER 3); H3 DNA; T4 ligase; 10X T4 ligase buffer; 10% PEG; Millipore nitrocellulose filters

METHODS:

  1. Place H3 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 a ligation reaction as described below:

    3. Ligation reaction

    20 ng vector DNA

    10 µl 10X T4 ligase buffer

    2 µl T4 ligase (i.e., 6 units)

    200 ng of H3 DNA

    MBG water to give a final reaction volume of 100 µl

  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. To create a 16ºC environment, a standard water bath can be placed in a refrigerator and the temperature knob can be adjusted until a stable temperature of 16ºC is attained. Do not incubate ligation reactions for > 20 hrs.
  5. Place ligation reaction tubes in a 65ºC water bath for 20-30 min to "heat kill" the enzyme.
  6. Ligated DNA must be desalted before it can be used in transformation. Desalting can be performed quite easily using Millipore 0.025 µm filters. Add 30 ml of sterile 10% PEG to a 90 mm Petri plate. Place a Millipore nitrocellulose filter (with its shiny-side facing up) on the PEG surface of the plate. Using a pipettor with a large-orifice pipet tip, transfer the contents of the ligation reaction tube to the center of the Millipore filter (FIGURE 4.1). Place a lid on the Petri plate. Allow the DNA sample(s) to desalt for 90 minutes.
  7. Using a pipettor and large-orifice pipet tips, transfer the desalted ligation reaction into a 1.5 ml microcentrifuge tube (i.e., pool the ligation reactions).

    8. ­ Note 4.1: For each filter, some of the ligation solution will be so closely associated with the filter surface that it will not be recovered.

    ­ Note 4.2: 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 4.3: Ligated DNA is stable at 4°C for at least 5 days.

 

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