Cloneamp polymerase

Genomic DNA was extracted from the legs of ten adult Thermobia specimens using the DNeasy Blood and Tissue Kit (Qiagen). External primers designed for contig GASN01030700.1 were used to amplify the full gene (from start to stop codon) using genomic DNA as template and CloneAmp polymerase (Clontech) via nested PCR. The product, with estimated size of 4.5 kbp, was cloned with the StrataClone Blunt PCR Cloning Kit (Stratagene). The gene structure was then determined through Sanger sequencing using internal primers. Intron/exon boundaries were identified by comparing the full gene sequence with the coding sequence from the cDNA.

The pCDH-MND-OsTIR1(F74G)-P2A-EGFP^AID2-EF1a-RFP construct was made by Gibson assembly [53 (link)]. The OsTIR1(F74G)-P2A-EGFP^AID2 fragment was amplified from the pAAV-hSyn-OsTIR1(F74G) vector (Addgene, 140,730) and cloned into the EcoR1 site of pCDH-EF1a-RFP. After cloning, the CMV promoter was replaced by Gibson assembly with an MND promoter to overcome promoter silencing [54 (link)]. The inducible CTCF series was created by cloning the WT CTCF and CTCF dZF1, dZF10, and dRBR (− 120 bps following ZF11) into a Tet-on-3G-inducible vector. Primers were designed to amplify CTCF from a pT2K-CTCF-HA construct we had previously cloned. The mutants were generated by designing primers to amplify two fragments that flanked either ZF1, ZF10, or the RBR and would exclude these features after Gibson assembly. Snapgene software was used to design all primers used for cloning. The PCR reactions to amplify all products for cloning were performed using CloneAmp polymerase (Clontech) and the cycling parameters were 98 °C for 5 min, followed by 40 cycles of 98 °C for 15 s, 55 °C for 20 s, and 72 °C for 20 s. Gibson assembly reaction mix was made as previously described [53 (link)], and all reactions were carried out at 50 °C for 20 min. All primer information was included in the Additional file 2: Table S1.