Journal: BMC Biotechnology
Article Title: FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method
Figure Lengend Snippet: Optimization of cloning conditions . (A) PCR amplification of a target cDNA (human nAChR α9 subunit) and the pGEMHE vector using different DNA polymerases: Pfu Turbo, PfuUltra and Phusion. (B) Comparison of number of colonies grown on the plates after transformation. Three different vector-to-insert ratios (1:1, 1:2, and 1:4) during Dpn I digestion and three amounts of vector-insert mixtures (2, 4, and 8 μl) for transformation were tested. See text for details. (C) Clone validation by PCR using GoTaq DNA polymerase. Lanes 1 to 12: target clones to be validated; Lane 13: 1 Kb plus DNA ladder; Lane 14: pGEMHE vector control; Lane 15: negative control using pCR4-TOPO-α9 parent plasmid. (D) Clone validation by restriction digestion to exclude unusual constructs. Lane 1: 1 Kb plus DNA ladder, Lanes 2-11: target clones double digested with Kpn I and Nhe I. Note that this digestion resulted in a pGEM vector and an insert with α9 nAChR plus the 5'UTR and 3'UTR of Xenopus β-globin.
Article Snippet: The PCR reaction components were: 50 μl total volume, 0.5 μl Phusion DNA polymerase (New England Biolabs, Ipswich, MA), or 0.8 μl Pfu Turbo, or PfuUltra DNA polymerase (Agilent Technologies, Inc, Santa Clara, CA), 5 μl 10× buffer; 5 μl of 2.5 mM dNTPs; 10 ng of plasmid DNA template; 5 pmol of each primer.
Techniques: Clone Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Transformation Assay, Negative Control, Construct