CHAPTER 5

Transformation is the process by which recombinant DNA is taken up by competent bacteria to produce BAC clones. Transformation is an essential step in all DNA cloning protocols.

In order to determine if the vector is suitable for future ligations and transformation, the ligation product produced in CHAPTER 4 is used to transform competent cells. If lambda DNA fragments have been ligated into the polycloning sites of many of the BAC vector molecules, transformation using the test ligation should produce white colonies due to alpha-complementation.

In transformation, competent bacterial cells are mixed with ligate. The resulting mixture(s) is exposed to an electric shock that temporarily creates holes in the plasma membranes of the bacterial cells. This allows BACs to be taken up by the bacteria.

There are currently a variety of commercially available electroporation devices. Because these devices differ in design and operation instructions, researchers should consult manufacturer's instructions to obtain best results. The Gibco BRL Cell-Porator^TM is a commonly used electroporator. Likewise, it is specifically recommended for use with DH10B cells. Detailed instructions regarding operation of the Gibco BRL Cell-Porator are given in APPENDIX A.

SUPPLIES, EQUIPMENT, AND REAGENTS (see CHAPTER 2 for details): 70% ethanol; ElectroMax® DH10B^TM competent cells; ligated DNA produced in CHAPTER 4; SOC; X/I/C Petri plates; glass plating rod; electroporator with cuvettes

METHODS:

1. Place 2.0 µl of the ligated DNA into four 0.65 ml microcentrifuge tubes. Place the remaining ligated DNA in the refrigerator.
2. Add 20 µl of competent cells to each of the four microcentrifuge tubes. Tap the tubes gently to mix contents. Place the tubes on ice.
3. Using a pipettor equipped with a standard 200 µl tip, transfer the mixture from one of the tubes into an electroporator cuvette. Make sure that there are no bubbles in the ligate/bacterial solution. Load each of the remaining mixtures into its own cuvette.
4. Use an electroporator to apply a shock to the bacteria in each cuvette. Because the lambda DNA fragments are relatively small compared to the fragments that will be used in BAC library construction, a relatively high voltage of 390-400 should be used.

­ Note 5.1: Be careful not to shock cells in a cuvette more than once.

5. Place the cuvettes on ice.
6. In a laminar-flow hood, place 0.5 ml of sterile SOC into four sterile 15 ml culture tubes (equipped with sterile caps).
7. Using a pipettor, carefully transfer the contents of each cuvette into the media in one of the culture tubes. Label the tubes I, II, III, and IV, place a cap on each culture tube, and incubate tubes at 37ºC with 250 rpm agitation for exactly one hour.
8. Immediately after placing culture tubes in the incubator, take four X/I/C Petri plates out of the refrigerator. Allow the plates to warm to room temperature, but keep them in a cabinet or wrapped in foil to prevent exposure to light.
9. Remove the "one-hour" culture tubes from the incubator. Transfer culture tubes and X/I/C Petri plates to a laminar flow hood equipped with a Bunsen burner or alcohol lamp.
10. Flame-sterilize a glass-plating rod as shown in FIGURE 5.1. Allow the rod to cool for approximately one minute.
11. Place 50 µl of culture I onto the agar of one of the Petri plates. Use the plating rod to spread the culture over the entire agar surface (FIGURE 5.2). Continue moving the rod across the agar until the rod begins to glide with less fluidity, i.e., the friction between the rod and the agar increases. This indicates that the bacterial culture has been absorbed into the agar.
12. Replace the lid and turn the plate upside-down. On the bottom of the plate write "I".
13. Prepare a plate for each of the three remaining test transformation reactions (i.e., II, III, and IV) as described in steps 10-12 above.
14. Place the four test plates in a 37ºC incubator overnight. Clones should appear within 15 hours of incubation and should reach a diameter of 1-2 mm by 20 hours.
15. Determine the average number of colonies per plate, the percentage of clones that are white, and the percentage of clones that are blue. Based on the fact that 50 µl of culture was spread on each plate, determine the number of CM-resistant bacteria (colony forming units = cfu) per microliter of liquid culture. For example, if there is an average of 135 colonies per plate, the one-hour cultures possess roughly (135 cfu/plate ÷ 50 µl of one-hour culture/plate =) 2.7 cfu/µl.

• A good transformation is characterized by > 100 cfu/plate (> 2 cfu/µl) and no more than 20% blue colonies. If the titer is < 0.5 cfu/µl, it is possible that the ratio of insert DNA to vector DNA in the ligation reactions was too high. A high percentage of blue colonies suggests problems during ligation and/or dephosphorylation.
• If the titer is > 0.5 cfu/µl and over 50% of the colonies are white, it is generally worth doing minipreps and NotI digestions using BACs isolated from white colonies on the test plates (see CHAPTER 6).

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