Zero blunt topo pcr cloning kit for sequencing

Pure solutions of viral particles from Exact Diagnostics were created for each virus from the concentrations of 10⁵ to 10¹ (cp/mL or IU/mL for CMV). To do this, the stock solution of 10⁶ was diluted 1:10 in TE Buffer so that a 200 µL solution for each desired concentration was made. The extraction was performed in the same method as described above using the Zymo Quick-DNA Microprep Plus (Zymo Research). After DNA was eluted, the same PCR was performed as mentioned in the previous section. The results of this PCR were used to import a reference curve into the PCR runs testing the enriched vs. non-enriched samples at each concentration. This allowed for the software to estimate the concentration of each sample run based on the set standard concentration of the pure solutions used in the run to create the reference curve. The VZV reference curve was constructed using a VZV plasmid which was cloned using the Zero Blunt™ TOPO™ PCR Cloning Kit for Sequencing (Thermo Fisher).

To determine the 5′-terminal sequence of traA mRNA, cDNA was synthesized using total RNA of Anc(C) as the template, SuperScript®; III reverse transcriptase (Life Technologies, Carlsbad, CA, USA), and traA_r primer. The 5′-phosphorylated DNA linker 5PpACYC_rev was ligated at the 3′-terminus of the first strand cDNA with T4 RNA ligase 1 (New England Biolabs). PCR was performed using the resultant cDNA as the template, PrimeSTAR®; HS DNA polymerase (Takara Bio Inc., Shiga, Japan), and the primers pACYC_rev2 and traA_r2. To determine the 3′-terminal sequence of traA mRNA, universal miRNA cloning linker (5′-rAppCTGTAGGCACCATCAAT-NH2-3′; New England Biolabs) was ligated with the 3′-terminus of total RNA of Anc(C) using T4 RNA ligase 1 (New England Biolabs). The first strand cDNA was synthesized using the primer Linker_r and SuperScript®; III reverse transcriptase (Life Technologies), and then purified cDNA was subjected to PCR using the primers Linker_r and traA_f. Three and two bands of PCR products for 5′- and 3′-terminal sequence determination, respectively, were sliced from the gel and subcloned using a Zero Blunt®; TOPO®; PCR Cloning Kit for Sequencing (Life Technologies). Three to six clones were randomly picked and sequenced by the dideoxynucleotide chain termination sequencing method.

For 3′RACE the total RNA was DNase treated (Promega, WI) and oligo(dT₂₅)T7 primers were used for reverse transcription. The 3′RACE reactions were performed using Phusion master Mix (ThermoFisher Scientific) and reverse primers as previously described⁴⁰ . To simultaneously detect all forms of ABCC1 3′UTRs, we used the nested primers within the open reading frame of ABCC1. The sequence of the forward primer used for the first round of PCR was 5′-GACCTCCGCTTCAAGATCAC-3′ and for the second PCR was 5′-GAATGAACCTGGACCCATTCA-3′. After gel purification using the gel extraction wizard SV gel and PCR cleanup system (Promega), the second round PCR products were cloned using Zero Blunt TOPO PCR Cloning Kit for Sequencing (Life Technologies) and verified by Sanger sequencing (Lonestar Labs).

The DNA products generated by 5′ RLM-RACE and 3′ RACE were each resolved by electrophoresis in a 1% agarose gel and purified using the MinElute gel extraction kit (Qiagen, Valencia, CA). The gel-purified 5′ RLM-RACE- and 3′ RACE-derived DNA were cloned using the Zero blunt TOPO PCR cloning kit for sequencing (Life Technologies) and the pGem T-Easy vector (Promega, Madison, WI), respectively, according to the manufacturer’s instructions, and transformed into E. coli (DH5α). Multiple clones were selected and sequenced to determine the nt identities of the cloned DNA. DNA sequencing was conducted in the Genomics Core of the Institute for Integrative Genome Biology (IIGB) at the University of California, Riverside. Selected CTV sequences with the T30 genotype (GenBank accession nos. KC517489, KC517490, KC517491, AF260651, Y18420, KC748391 and KU578007) and T36 genotype (GenBank accession nos. KC517485, KC517487, EU937521 and AY170468) were used for nucleotides comparison using the Clustal Omega program (https://www.ebi.ac.uk/). The identities of the most commonly seen nt variants as well as the frequency with which these variant nts appeared in the cloned DNA are shown in Tables 1 to 2.