vector보다 insert가 30배 크다면 BAC cloning 방법을 사용하세요.
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 microfuge tubes as follows:
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 microfuge 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 onethird 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.