Pcr clean up kit

The coding sequences of Arabidopsis thaliana NPR1 (AT1G64280), NPR4 (AT4G19660) and NIMIN2 (AT3G25882) were amplified by polymerase chain reaction from an Arabidopsis cDNA library, and sub-cloned into the DH5α vector with different N-terminally fused tags and a tobacco etch virus (TEV)-cleavage site. The specific amino acid changes for the NPR1 and NPR4 point mutations were generated using the QuikChange II site-directed mutagenesis kit (Agilent). Glutathione-S-transferase (GST)-fused NPR4 coding sequence (CDS) was subcloned into the pFastBac vector and transformed to E. coli DH10Bac for making baculovirus for protein expression in insect cells. Protein domain swaps were generated by amplifying the desired regions of each CDS with primers designed to create overlapping sequences for each fragment. The DNA fragments were amplified in separate PCR reactions, processed with either a PCR clean-up kit or gel extraction kit (Bio Basic Inc.), and the desired fragments were fused by PCR using gene-specific forward and reverse primers containing attB1 and attB2 Gateway™ recombination sequences, respectively. All CDSs were recombined into pDONR207 or pDONR221 and subsequent expression vectors using the Gateway™ technology and sequenced to confirm accuracy.