Promega Notes Cloning Blunt-End DNA Fragment Into the pGEM#174;-strongTstrong.pdfVIP

Promega Notes Magazine Number 62, 1997, p. 15 Cloning Blunt-End DNA Fragments Into the pGEM®-T Vector Systems By Gary Kobs Promega Corporation Blunt-end DNA fragments can be ligated into Promegas T- Vectors if they are first tailed using dATP and Taq DNA Polymerase(a) (1). In this article, the results of two different A-tailing protocols were evaluated using a pGEM®-T(b,c) Easy Vector System. A standard A-tailing protocol was compared to an abbreviated alternative protocol using both PCR products generated by thermostable DNA polymerases that have proofreading activity, and modified blunt -end DNA fragments generated by restriction digestion. The efficiency of cloning long PCR(d)fragments into the pGEM ®-T Vector Systems was also evaluated. (a)Some applications in which this product may be used are covered by patents issued and applicable in certain countries. Because purchase of this product does not include a license to perform any patented application, users of the product may be required to obtain a patent license depending upon the particular application and country in which the product is used. For more specific information, please contact Promega. (b)Licensed under one or both of U.S. Pat. No. 5,487,993 and European Pat. No. 0 550 693. (c) U.S. Pat. No. 4,766,072 has been issued to Promega Corporation for transcription vectors having two different bacteriophage RNA polymerase promoter sequences separated by a series of unique restriction sites into which foreign DNA can be inserted. (d)The PCR process is covered by patents issued and applicable in certain countries. Promega does not encourage or support the unauthorized or unlicensed use of the PCR process. Introduction Certain thermostable polymerases, including Taq, Tfl and Tth DNA Polymerase(a), add a single nucleotide, generally adenine, to the 3´- ends of amplified DNA fragments (2,3). Promegas T-Vector Systems (the pGEM®-T, pGEM®-T Easy and pTARGETTM(b) Vectors) are convenient sy