Aging Aging

Differentially Expressed Genes 109Dephosphorylated, restricted plasmid (50 ng)/phosphorylated, blunt ended fragmentin a ratio 1:3 to 1:51× Ligase buffer1 µL of T4-ligase (5 U/µL)(total volume 20–40 µL)The probe can then be incubated at 4°C overnight or at 16–20°C for 3–4 h.The probes can be stored at –20°C.3.9.5. Preparation of CaCl 2 competent cells for transformationThe E. coli cells are treated following the CaCl 2 procedure from Mandeland Higa (13).1. 50 mL of LB medium are inoculated with 5 mL of an overnight preculture ofthe E. coli strain. The culture is then incubated at 37°C to an OD (600 nm) from0.3 to 0.4.2. The cells are centrifuged (2500g, 10 min, 4°C) and the pellet is resuspended in100 mL of ice-cold 50 mM CaCl 2 solution.3. The cells are centrifuged again (only 1800g, 4°C).4. The pellet is resuspended carefully in 20 mL of CaCl 2 and incubated for 20 minon ice.5. The cells are centrifuged again (1800g, 4°C) and the pellet is then carefully resuspendedin 10 mL of ice-cold CaCl 2 solution. In addition, glycerine is added to afinal concentration of 20%. The probes should be divided in aliquots of 200 µL inmicrofuge tubes and then they can be stored at –80°C for several months.3.9.6. Transformation1. Prior to transformation, the ligation reaction mixture is filled with ddH 2 O to afinal volume of 100 µL.2. This mixture is added to a 200-µL aliquot of not completely thawed competentbacteria. The probe is then incubated 30 min on ice.3. After that the bacteria are heat shocked for exactly 45 s at 42°C and 1 mL of 37°Cwarm LB medium is added at once.4. The bacteria are incubated for 30–40 min. (They should have the possibility todivide once the plasmid incorporation is stabilized.)5. Then aliquots of, e.g., 50, 100, and 200 µL are plated onto an LB agar plate withappropriate antibiotics or other selection markers (e.g., β-galactosidase).6. The plates are incubated at 37°C overnight and can be stored at 4°C for a fewdays. Positive clones can be inoculated into LB medium with a suitable antibioticand after an overnight incubation at 37°C (shake) the plasmids can be isolated forfurther control.3.9.7. Plasmid Isolation by Alkaline LysisFor plasmid isolation, many reaction kits are available that can result inextremely pure plasmids. These are required, for example, for sequencing but