Aging Aging

108 Engel, Adibzadeh, and Pawelec3.9.2. 5'-Phosphorylation of the FragmentsAfter the blunting of the fragments the 5'-ends have to be phosphorylatedbefore they can be cloned into a plasmid vector. The enzyme T4-polynucleotidekinase (PNK) is employed for this purpose.1. Add to the probe of 3.9.1:2 µL of ATP (10 mM)1 µL10× PNK buffer1 µL of PNK(final volume: 20 µL)An incubation step of 30 min at 37°C is performed.2. At this stage the PNK can be inactivated for 10 min at 70°C and the probes can bestored at –20°C.3. Before cloning the probes have to be purified by agarose gel electrophoresis. Thefragments of interest can then be separated via the gel from additionally amplifiedfragments and primer dimers from the reamplification reaction.3.9.3. Preparation of the Plasmid Vector1. The plasmid vector (e.g., pUC18, pUC19, Bluescript, or any other vector) isdigested with a blunt ending restriction enzyme that cuts the plasmid vector intothe multiple cloning site (MCS; i.e., SmaI) (see also Note 6).The restriction conditions depend on the enzyme used and should be followedaccording the product information. The volume should be between 20 and 50 µLand 2 U of the enzyme per microgram DNA should be added. After the incubationthe enzyme has to be inactivated. It is mostly possible to heat inactivate therestriction enzymes, but if this should not be the case (see the product information)an agarose gel purification or ethanol precipitation has to be done. The purificationof the vector from the restriction enzyme and the buffers is also necessaryprior to the following dephosphorylation reaction.2. To avoid the religation of the vector in the ligation reaction, it has to be dephosphorylatedwith an alkaline phosphatase. The dephosphorylation reaction is donewith 0.04 U alkaline phosphatase/µg DNA in 1× phosphatase buffer (provided bythe manufacturer) at 37°C for 60 min. After the incubation the vector has to bepurified in an agarose gel and eluted with a suitable method.3.9.4. LigationFor the ligation reaction there are many kits provided by several companies.We have had good experience with the Rapid Ligation Kit (Boehringer, Mannheim).This is a method for cloning blunt ended fragments into plasmid vectors.But of course any other ligation method, without using a kit, could besuccessful (see also Notes 8 and 9).A ligation procedure without using a kit requires the following reaction mix: