Pcr cleanup kit

In order to make pSC1, the gtr6-hyg chimeric cassette was inserted in the middle of the lacZ gene of puc19, where all of the gene except of 5’ end 32 bases, was deleted. However, the gtr6 gene in pSC1 was devoid of its promoter and was not inducible. Additionally, as a small portion of the 5’ end of the lacZ gene remained, the gtr6 gene could not be induced by the lac promoter either. Hence, we decided to delete the 5’ end fragment of lacZ from the gtr6 upstream region and clone the 192 base pair long indigenous promoter region of gtr6 upstream of the gene itself thus creating pSC1a. The plasmid pSC1 (S2E Fig) and the gtr6 indigenous promoter sequence (192 bp) were PCR amplified (S3 Text). The linearized plasmid PCR product was purified with NEB PCR clean up kit using manufacturer’s protocol while the promoter region PCR product was gel purified by NEB Gel purification kit following manufacturer’s protocol. The linear fragments were subjected to Gibson cloning using NEB Gibson Cloning kit following manufacturer’s protocol and was transformed in to NEB 5α Competent E. coli cells. The recombinant clones were selected on LB Hygromycin (150μg/mL) agar plates. Putative clones were grown overnight in 5mL LB Hygromycin (150μg/mL) broth and 1uL was used to perform colony PCR with primers Gtr6-Hyg Internal 5&3 as described previously.

The amplified PCR products were purified using a PCR Cleanup kit (NEB, USA), following the manufacturer’s instructions, and sent to Macrogen (The Netherlands) for sequencing. The quality of sequences was verified using the software BioEdit 7.0.9.0 (47 ). The BLASTn program was used to identify closely related species in the NCBI database. Pairwise and multiple-sequence alignments were carried out with CLUSTALW, and phylogenetic trees were constructed by means of the maximum likelihood statistical method using MEGA7 software (48 (link)). The evolutionary history was inferred by means of the maximum likelihood method based on the Kimura 2-parameter model (49 (link)). The robustness of branching was estimated using 1,000 bootstrap replicates (50 (link)).

pcDNA4 T7 c-JUN and pcDNA4 T7ACTB plasmids used for in vitro transcription were provided by Dr. Amy Lee (Brandeis University, Waltham, MA)^{22 (link),23 (link)}. The c-JUN template was constructed by amplifying the ORF and 3′ untranslated region from human cDNA and joined together downstream of a T7 promoter by Gibson assembly into pcDNA4. After added the T7 promoter to ACTB by PCR amplification the fragment was inserted into pcDNA4.
CDK12 and MMP1 templates were generated by PCR amplification and addition of a T7 RNA polymerase promoter with high-fidelity DNA polymerase (NEB#M0491). The design primers containing the T7 promoter followed by sequences of interest are:
5′-CATATGTAATACGACTCACTATAGGATGCCCAATTCAGAGAGA-3′ and
5′-CGCGGCCGCAGTAAGGAACTCCTCTC-3′ for CDK12, and
5′-CATATGTAATACGACTCACTATAGGATGCACAGCTTTCCTCC-3′ and
5′-GGCGGCCGCAATTTTTCCTGCAGTTGAACC-3′ for MMP1.
Prior to its use as a template for in vitro transcription, the PCR products were analyzed by agarose-gel electrophoresis and purify by PCR Cleanup Kit (NEB#T1030).