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Unformatted text preview: Subcloning: Preparation of Vector In this step of the ctoning experimenq you will prepare the vector (plasmid) for ligation To prepare the vector, it will be digested with restriction enzymes and dephosphorylated. The vector will be dephosphorylated to reduce the background intramoleorlar ligations. There are several sources of alkaline phosphatase that are commercially available: bacterial (BAP), calf intestinal (CP), shrimp (SAP) and *Antarctic'. Bacterial alkaline phosphatase is very heat stable, which is a draw back when one wishes to inactivate the enzyme prior to the ligation reaction CIP and SAP can be readily inactivatod by heating at 65C for 30 minutes. "Antarctic" is the most heat labile: 5 minutes at 65C will completely inactivate the activity. You will also be givgn an agarose gel to check the vector and insert by electrophoresis. Workinlnirs l. Add the following to the microfuge tube which contains l0 ug ofpBSK (providd by your TAdtJAs): 6.5 pl rIzO 2 tLl l0x restriction buffer 0.5 pl Fr,oKI 0.5 pl HitKIIfl Note: Always add enzyme last to reaction and always ensure that the enzyme is less than l@/o of total volume. Enzrymes are stored in glycero[ which can inhibit the activity of the enz)rme. Always keep enzymes cold: either on ice or in the freezer. 2. Tap to mix Centrifuge 30 seconds (max speed)....
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This note was uploaded on 07/13/2008 for the course MCDB 104 taught by Professor Lee during the Summer '08 term at UCLA.
- Summer '08