Pcr xl 2 topo vector

5′ Rapid amplification of cDNA ends (5′ RACE) was performed as described previously (18 (link)), but omitting the 5′-triphosphate removal step (by RNA 5′ pyrophosphohydrolase treatment) to exclusively detect processed RNA molecules. Briefly, total RNA was isolated as described above. A specific RNA 5′ adapter (Table 2) was then ligated to the RNA. After phenol/chloroform extraction and ethanol precipitation, the RNA was subjected to reverse transcription using gene-specific primers (for metI: FW200, for metF: FW161) (Table 2), followed by PCR amplification (for metI: Sa_lgsm02-Lext-Rev and 5′ RACE RNA adapter primer, for metF: FW149 and 5′ RACE RNA adapter primer) (Table 2) and cloning of the PCR products into pCR-XL-2-TOPO vector (Thermo Fisher Scientific, #K8050) and transformation into E. coli DC10B. 5′- ends were analyzed by nucleotide sequencing and mapped to the met operon of S. aureus strain Newman as reference sequence using the CLC Main Workbench analysis tool (Version 6.6.1; www.clcbio.com).

Genotyping PCR was performed using genomic DNA extracted from the toe clipping with the primers listed in Supplemental Materials. For Sanger sequencing, PCR products were first gel-purified and cloned into the pCRII Topo vector (Thermo Fisher Scientific K460001) and sequenced with T7 or SP6 primers. Long-range PCR was performed using LongAmp Hot Start Taq 2X Master Mix (NEB M0533S) to examine genomic DNA that was purified by a Monarch Genomic DNA Purification kit (NEB T3010). The large amplicons were gel-purified and cloned into the pCR-XL-2-TOPO vector (Thermo Fisher Scientific K8050-10). The top off-targeting sites were predicted by Cas-OFFinder (Bae et al. 2014 (link)).