CHAPTER 3

Vector Isolation

BAC cloning requires successful isolation and preparation of the BAC vector. Poorly prepared vector DNA is one of the primary causes of failure in BAC library construction, and thus vector preparation should be performed with the utmost care.

Vector preparation includes amplification of the BAC vector via host cell propagation, isolation of the vector DNA, restriction digest of the vector to produce cohesive termini (sticky ends), and dephosphorylation. Dephosphorylation removes the 5'-phosphate group from the last nucleotide on each sticky end. The absence of 5'-phosphate groups prevents termini from being ligated together but allows ligation between dephosphorylated termini and complementary cohesive termini on DNA molecules that have not been dephosphorylated (i.e., insert DNA) (Sambrook et al. 1989).

Phosphatase, the enzyme used to dephosphorylate the vector, is extremely "aggressive". If the vector DNA is "over-exposed" to the phosphatase enzyme, cohesive termini will not only be dephosphorylated but will be effectively destroyed. Thus exposure of the vector to phosphatase must be limited. One result of limiting vector dephosphorylation is that not all of the vector molecules will be dephosphorylated. In the presence of T4 ligase, vector molecules that have not been dephosphorylated can re-circularize or interact with other phosphorylated and/or dephosphorylated vector molecules to form concatemers. If these recircularized or concatemerized vector molecules are around during transformation, they will result in blue colonies (non-recombinant clones) or false positive colonies, respectively. To get rid of most of the vector molecules that have not been dephosphorylated prior to ligation with insert DNA, the vector DNA can be pretreated with T4 ligase in a "self-ligation" reaction. Vector molecules possessing one or more phosphorylated ends will either recircularize or be ligated to other vector molecules with one or more phosphorylated termini. Concatemers and re-circularized BAC vectors subsequently can be separated from linearized vector molecules by gel electrophoresis. Self-ligation followed by electrophoretic isolation of dephosphorylated vector greatly improves the results of transformation.

SUPPLIES, EQUIPMENT, AND REAGENTS (see CHAPTER 2 for details): Qiagen Large-Construct Kit; pBeloBAC11 vector in DH10B; LB^+CM; HindIII with 10X buffer and 100X BSA; T4 ligase with 10X ligase buffer; HK phosphatase with 10X phosphatase buffer and 0.1 M CaCl₂; 1X TAE; agarose; H3 DNA; blue juice; ethidium bromide; UV light box equipped with a camera or image-capture system; 1X uncut lambda DNA

METHODS:

1. Isolate BAC vector from DH10B bacterial culture (LB^+CM) using a Qiagen Large-Construct Kit. Perform the isolation according to manufacturer’s instructions.
2. Set up a restriction digest in a 0.65 ml microcentrifuge tube using 10 µg of the vector as follows:

3. Mix the contents of the microcentrifuge tube by tapping on the tube, and place the reaction at 37ºC for 2 hours.
4. Place the tube in a 65ºC water bath for 20 minutes. This effectively destroys the HindIII. Allow the tube to cool at room temperature for 10 minutes and briefly spin the tube in a microcentrifuge (10,000 rpm for 30 sec).
5. To the reaction tube add 5.0 µl HK phosphatase, 5.0 µl of 0.1 M CaCl₂, 2.5 µl of 10X phosphatase buffer, and 12.5 µl MBG water.
6. Gently mix the contents of the tube, and place the tube in an incubator at 30ºC for 2 hours or in a water bath at 30ºC for 90 min.
7. Destroy phosphatase activity by placing the reaction at 65ºC for 20 min. Allow the reaction to cool at room temperature for 10 min and briefly spin the tube in a microcentrifuge.
8. To the tube add 12.5 µl 10X ligase buffer, 3.0 µl of T4 ligase, and 9.5 µl of MBG water. Mix gently by tapping on the tube.
9. Place the tube at room temperature for 2 hours or leave it overnight (12 to 16 hours) at 16ºC.
10. Heat-kill the T4 ligase by placing the reaction tube at 65ºC for 20 min.
11. Prepare a small 0.8 % w/v agarose gel in 1X TAE buffer. After the gel has solidified, remove the comb. Using a scalpel, carefully remove the agarose between several of the comb-made wells to produce a slot well (FIGURE 3.1a). Make the slot well large enough that it can accommodate roughly 150 µl of liquid (but not much more).

­ Note 3.3: We commonly cut agarose gels with a scalpel or razor blade. However, some prefer a coverglass arguing that nucleases may be activated by metal ions from the scalpel/razor blade.

12. Place a thin layer of melted 0.8 % w/v agarose in the bottom of the slot well. Allow the agarose to solidify.
13. Place the gel in an appropriate mini-gel electrophoresis chamber. Add 1X TAE buffer to the electorphoresis chamber until the gel is completely covered with buffer.
14. Add 13 µl of blue juice to the tube containing the dephosphorylated, self-ligated vector solution. Gently mix the tube’s contents, and spin the tube in a microcentrifuge at 13,000 rpm for 30 sec.
15. Transfer the contents of the tube into the slot well (FIGURE 3.1a).
16. Place a mixture consisting of 2 µl of H3 DNA, 5 µl of MBG water, and 2 µl of blue juice in each of the wells flanking the slot well (FIGURE 3.1a).
17. Run the mini-gel for 5 hours at 3.7 v/cm.
18. After electrophoresis, cut the gel with a scalpel as shown in FIGURE 3.1b. Use a plastic ruler as a "straight-edge" to guide the movement of the scalpel through the gel.
19. Gently remove the two flanking gel pieces from the underlying casting tray, and place them in a photographic developing tray. Add enough distilled water that the gel pieces can move freely. Add one drop of ethidium bromide (10 mg/ml) and place the tray on a shaker table. Set the speed of the shaker table so no spilling of ethidium bromide solution occurs. Allow the gel to stain for 20 min. Carefully pour off the ethidium bromide solution (into an appropriate hazardous waste container) and add distilled water to the tray. Place the tray back on the shaker table and allow the gel to "destain" for 20 min.
20. Align the two flanking gel pieces on a UV light box. Turn on the light box.

­ Note 3.4:Always wear eye and face protection when using the UV light box!

21. The dephosphorylated vector should appear at 7.5 kb. Linear concatemeric DNA may be visible at molecular weights > 7.5 kb. Circularized vector and vector concatemers will migrate more slowly than linear vector molecules and thus will be found at molecular weights > 7.5 kb. Using a scalpel, make horizontal incisions in the flanking gel pieces to delimit the 7.5 kb vector band (FIGURE 3.1c).
22. Turn off the UV light box. On a piece of clean plastic wrap on a workbench, assemble the two flanking gel pieces and the unstained central gel piece. Using the incisions on the flanking pieces as guides, cut the 7.5 kb band from the unstained portion of the gel (FIGURE 3.1d). Once the unstained 7.5 kb vector band has been removed, the remaining unstained gel pieces can be stained, and the gel pieces (with the exception of the unstained vector DNA) can be reassembled and photographed for documentation purposes.

­ Note 3.5: Never expose the gel piece containing the unstained vector to UV light as this will nick the vector DNA making it unusable in cloning!

23. Cut the gel piece containing 7.5 kb vector DNA into 0.5 g pieces and distribute into 1.5 ml tubes. Freeze the tubes at –80ºC for 1 hour, and then immediately spin the tubes at 13,000 rpm in a microcentrifuge for 20 min at room temperature.
24. Using a pipet, carefully remove liquid (1X TAE containing dephosphorylated vector) from each tube. Place the liquid from all the tubes into a single microcentrifuge tube. It is not necessary to precipitate or desalt vector DNA prepared in this manner.
25. Prepare a 1% agarose submarine mini-gel in 1X TAE. In preparing the gel, use a comb with at least five teeth.
26. Place 0.5, 1.0, 2.0, and 4.0 µl of 1X uncut lambda DNA (i.e., 25 ng, 50 ng, 100 ng, and 200 ng) in separate 0.65 ml microcentrifuge tubes. Add 2.0 µl of blue juice to each.
27. Take a 2.5 µl aliquot from the tube containing the isolated vector and place the solution in a 0.65 ml microcentrifuge tube. Add 2.0 µl of blue juice.
28. Submerge the mini-gel in 1X TAE buffer in an appropriate mini-gel apparatus. Load the gel as shown in FIGURE 3.2. Run the gel at 100 v for 15-20 min. Stain and photograph the gel as described above. Based on comparison of the relative fluorescence in the sample and standard lanes, an estimate of the concentration of the vector DNA can be made.
29. Dilute the vector with sterile water to a final concentration of 10 ng/µl. Store the diluted vector at –20ºC in 25 µl aliquots in 0.5 ml microcentrifuge tubes. By aliquoting the vector DNA, you can avoid freeze/thaw cycles which will damage the vector. We recommend that vector which has been through more than 3 freeze/thaw cycles be discarded.

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