CHAPTER 15

If the results of the test transformation are satisfactory, a large-scale transformation is performed. The resulting transformants are plated onto X/I/C trays. White colonies picked from the trays will constitute the BAC library.

SUPPLIES, EQUIPMENT, AND REAGENTS (see CHAPTER 2 for details): 70% ethanol; ElectroMax® DH10B^TM competent cells; ligated DNA produced in CHAPTER 14; SOC; X/I/C trays; glass plating rod; electroporator with cuvettes (see APPENDIX A for detailed instructions regarding operation of the Gibco BRL Cell-Porator)

METHODS:

­ Note 15.1: Assuming the test transformation yielded satisfactory results, use the same electroporation device and the same instrument settings as were used in the test transformation (CHAPTER 14).

1. Place 2.0 µl aliquots of the ligated DNA into 1.5 ml microcentrifuge tubes. Because ligated DNA has a limited shelf life, it is generally best to go ahead and use all or most of the ligated DNA generated in CHAPTER 14. This means you may have 30 or more microcentrifuge tubes containing 2.0 µl of ligated DNA.
2. Add 20 µl of competent cells to each of the microcentrifuge tubes. Tap the tubes gently to mix contents. Place the tubes on ice.
3. For every six microcentrifuge tubes, place a 15 ml sterile culture tube (equipped with a sterile cap) in a test tube rack. Transfer the rack to a laminar-flow hood. Place 3.0 ml of SOC in each culture tube.
4. Using a pipettor equipped with a standard 200 µl tip, transfer the mixture from one of the microcentrifuge tubes into an electroporator cuvette. Make sure that there are no bubbles in the ligate/bacterial solution. Load as many additional cuvettes as possible.
5. Use an electroporator to apply a shock to the bacteria in each cuvette. Generally, a voltage of 320-330 is adequate.

­ Note 15.2: Be careful not to shock cells in a cuvette more than once.

6. Place the cuvettes on ice.
7. Once six cuvettes have been shocked, transfer the contents of these cuvettes into one of the culture tubes containing SOC. If need be, new transformation reactions can be loaded from microcentrifuge tubes into these emptied cuvettes.
8. Repeat steps 4-7 until all the transformation reactions have been transferred into culture tubes (six reactions per tube).
9. Place culture tubes in a 37ºC incubator shaker set at 250 rpm for exactly one hour.
10. Prepare a test plate for each "one-hour" culture tube as described in CHAPTER 5. Place the culture tubes in the refrigerator. Incubate the test plates at 37ºC overnight.
11. Based on the mean number of clones on the test plates, calculate a titer for the library (i.e., cfu/µl of "one-hour" culture. Additionally, calculate how many microliters of one-hour culture constitute 2000 cfu. For example, if the average number of colonies on each plate is 150 and each plate was innoculated with 50 µl of one-hour culture, the titer of the one-hour culture would be (150 clones/50 µl =) 3 cfu/µl. 2000 cfu would be present in (2000 cfu ÷ 3 cfu/µl =) 667 µl of overnight culture.

­ Note 15.3: If the trays are going to be picked by a QBot (see CHAPTER 16), a density of 2,000 clones per plate is desirable. If the clones are to be handpicked, a density of > 3000 clones per plate may be preferable.

12. The number of X/I/C plates one needs to prepare is based on the total volume of one-hour culture and the amount of one-hour culture constituting 2,000 cfu. For example, if you have ten culture tubes each containing 3 ml of culture, you have a total of 30 ml (30,000 µl) of one-hour culture. If 667 µl of one-hour culture contains 2,000 cfu, you should make up (30,000 µl of culture ÷ 667 µl =) 45 X/I/C trays. Prepare X/I/C trays as described in CHAPTER 2. Remember to UV sterilize the trays before pouring.
13. Remove the tubes containing one-hour culture from the refrigerator, and place them in the laminar-flow hood.
14. Flame-sterilize a glass-plating rod as shown in FIGURE 5.1. Allow the rod to cool for approximately one minute.
15. Place 2,000 cfu of one-hour culture onto the agar of one of the X/I/C trays. Use the plating rod to spread the culture over the entire agar surface (FIGURE 15.1). 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.

­ Note 15.4: In general, 0.5-0.8 ml of culture can be spread on a freshly prepared X/I/C tray without problem. However, smaller volumes may be absorbed into the agar before the bacteria can be thoroughly spread across the plate. This is especially likely if the plates have been stored for awhile before use. Likewise, larger volumes may take an excessively long time to soak into the agar. The former problem can be fixed by diluting the one-hour cultures with MBG water so that 2000 cfu are contained within 0.5-0.8 ml of fluid. The latter situation can be remedied by spinning the one-hour cultures at 650 x g for 20 minutes in a centrifuge, decanting the supernatants, and resuspending the pellets in just enough fresh SOC so that 2000 cfu are found in 0.75 ml of the mixture.

16. Replace the lid and turn the plate upside-down. On the side of the plate write information regarding the nature of the library (FIGURE 15.2).
17. Repeat steps 14-16 until all of the one-hour culture has been plated.
18. Place the X/I/C trays 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 (see FIGURE 15.3).
19. Select a few trays at random and use them to determine an estimate of the average number of colonies per plate, the percentage of clones that are white, and the percentage of clones that are blue.

• The trays should each contain between 2000-3000 colonies, and the vast majority of the clones should be white (e.g., FIGURE 15.3).

Return to CONTENTS

Go on to CHAPTER 16