Cloning Polymerase Chain Reaction Products Utilizing theT/A Overhang and a Kit
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The ever-expanding identification of new gene family members in recent years has depended in large part on the use of the polymerase chain reaction (PCR) technique. Typically PCR products may be cloned into a vector by cohesive- or blunt-end ligation. However, both cohesive- and blunt-end ligation traditionally require additional enzymatic manipulation and/or purification steps before the product may be cloned into the vector. Also, blunt-end ligation is a less efficient process than cohesive-end ligation (1). The Invitrogen (Carlsbad, CA) TA Cloning® Kit combines the efficiency of cohesive-end ligation with the ease of direct cloning of PCR products. A sample may be removed from the completed PCR reaction and transferred directly to the ligation reaction mixture. This procedure is based on the fact that Taq polymerase has a nontemplate dependent activity that adds a single deoxyadenosine to the 3′ ends of double-stranded DNA (2). The Invitrogen pCR™II vector is supplied as a linearized molecule with 3′ T overhangs on either side of the insert site. This allows cohesive-end ligation of the PCR product with the pCRII vector. In this chapter, I describe the steps that I followed in order to clone a 576-bp fragment of the rat N-cadherin gene into the pCRII vector. This plasmid was then used to generate RNA probes for use in a ribonuclease protection assay. Although much of the description of the ligation and transformation procedures is based on directions supplied by Invitrogen, some steps differ. Note that variations in protocol and vector have been introduced with more recent kits by Invitrogen. The time involved for this procedure is a total of 4 d: set up PCR reaction in the morning of the first day; after completion, set up ligation that afternoon; ligate overnight.
KeywordsPolymerase Chain Reaction Polymerase Chain Reaction Product Polymerase Chain Reaction Reaction Polymerase Chain Reaction Buffer Ligation Reaction
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