An improved inverse PCR protocol for the generation of T-vectors.

T-vectors have been widely used in molecular cloning [1–3]. Compared with common vectors, molecular cloning with T-vectors escapes the restriction endonuclease digestion process, decreases the time of DNA purification and thus is time-saving and cost-effective. There are two ways to produce T-vectors: (i) cutting the target vector with EcoRV and then adding a thymine residue to the 3′-blunt end with TaqDNA polymerase [4]; and (ii) inserting a DNA cassette containing XcmI or AhdI recognition sites at its both ends and then splitting the recombinant vector with them later [5–7]. However, only a small amount of the T-vector can be produced by these methods. What’s more, the residual undigested primary vector may cause false-positive clones. Therefore, T-vectors used in labs are usually bought from biotechnology companies providing with small amounts, thus limiting their application in conventional molecular cloning experiments. Here, we designed an improved inverse PCR protocol for the generation of T-vectors (Fig. 1). Briefly, an inverse PCR was performed with the target vector as a template with a pair of phosphorylated primers, then the PCR products were subject toDpnI treatment, agarose gel purification and further, the terminal deoxynucleotidyl transferase was applied to add a T to the 3′-blunt end of the PCR products which will ligate to the PCR products of the gene of interest with 3′-A overhangs. Afterwards, the ligation mixture was transformed into Escherichia coliDH5α competent cells, and a routine PCR was performed to calculate the positive rate of the transformed clones. The positive clones were further confirmed by DNA sequencing. Finally, we got at least 90% positive clones with our improved protocol. The main materials used in this study includes: the pcDNA3.0 vector, pcDNA3.0-FLAG vector containing human histone H4 cDNA, pcDNA3FLAG-CRBN vector all from our laboratory, PfuUltra II Fusion HS DNA polymerase from Stratagene (Cedar Creek, USA), the E. coli DH5α competent cells, DpnI, premix Taq, and T4 polynucleotide kinase (T4 PNK) fromTaKaRa Biotechnology (Dalian, China), ddTTP fromSigma-Aldrich (St Louis, USA),TaqDNApolymerasewith ThermoPol buffer, terminal deoxynucleotidyl transferase (TdT) from New England Biolabs (NEB; Massachusetts, USA), agarose gel DNA purification kit and PCR purification kit are all from Tiangen Biotech (Beijing, China). The sequence of the primer set for inverse PCR is as follows: forward primer 5′-TTCTATAGTGTCACCTAAATGCTAGA GC-3′, and reverse primer 5′-CCTATAGTGAGTCGTATTAATTT CGATAA-3′. The sequences of the primer pair for FLAGand human histone H4 fusion cDNA are as follows: forward primer 5′-CGGAATT CGCCACCATGGACTACAAGGACGACGA-3′; and reverse primer 5′-C GGGATCCACCGCCGAAACCATAAAGGG-3′; and the sequence of the primer pair for human CRBN cDNA is as follows: forward primer 5′-CCCAAGCTTATGGCCGGCGAAGGAGATC-3′; and reverse primer 5′-CGGGATCCTTACAAGCAAAGTATTACTTTGTC-3′. The T7 and SP6 primer pair was employed in routine PCR to evaluate the positive rate of the transformed clones. The DNA sequence of T7 primer is: 5′-TAATACGACTCACTATAGG-3′. The DNA sequence of SP6 primer is: 5′-ATTTAGGTGACACTATAGAA-3′. DNA sequencing was accomplished with the following primers: forward primer 5′-TTT CCAAAATGTCGTAACAACT-3′, and reverse primer 5′-AGACAATG CGATGCAATTTC-3′. All the above-mentioned primers were synthesized in Sangon Biotech Co., Ltd (Shanghai, China). To determined its usability, the T-vector generated from pcDNA3.0 vector was ligated to the coding sequence of FLAG tag and human histone H4 fusion protein (387 bp) as well as to the cDNA of human CRBN (1329 bp). The positive rate of the transformed clones was evaluated by routine PCR and the rightness of the insertion fragments was confirmed by DNA sequencing. Before the inverse PCR was performed, both the forward and reverse primers were phosphorylated in 20 μl reaction solution containing 5 μl primer (10 μM), 2 μl dNTP (10 mM), 2 μl T4 PNK buffer, 1 μl T4 PNK, and 10 μl distilled water. For inverse PCR, the following reagents were added into a 200 μl PCR tube: 10 ng pcDNA3.0 template plasmid, 400 μM dNTP, 0.8 μM forward and reverse primers respectively, 0.5 μl PfuUltra II Fusion HS DNA polymerase and distilled water was supplemented to 50 μl. Each PCR thermal cycle includes Acta Biochim Biophys Sin, 2015, 47(2), 142–144 doi: 10.1093/abbs/gmu118 Advance Access Publication Date: 23 December 2014 Lab Note