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Linear double-stranded DNAs (dsDNAs), such as PCR products flanked by short homologous sequences, can be used as recombination substrates with high efficiency.
PCR products flanked by the attB sites were recombined into the pDONR207 vector (Invitrogen) via a BP reaction to create the corresponding entry clones with attL sites.
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To further confirm whether both alleles of the Clock gene were modified in clone 17, clone 19, and clone 20, we performed TA cloning of the PCR products flanking the CRISPR/CAS9 cutting site.
Amplified products flanking the region to be deleted were purified using the QIAquick PCR purification kit (Qiagen) and digested by HindIII then subsequently ligated to one another.
Significant number of archaeal and bacterial genomes encode STYKs with regions most similar to ubiB gene products flanking the catalytic kinase domain.
One of the most popular locations at the event was an in-house medical marijuana dispensary stocked with Chong's Choice product (flanked by racks of brand T-shirts and bowls of give-away stickers, naturally) accessible to anyone who was able to present the proper legal paperwork.
The pHELLSGATE vector was designed such that a single PCR product flanked by att sites is recombined simultaneously to form the two arms of the palindrome.
A plasmid containing wild-type or mutant pie-1 sequence was used as a template to generate a left-arm PCR product flanked by BsiWI and NheI restriction sites and a right-arm PCR product flanked by NheI and NgoMIV restriction sites.
The amplified 1356 bp DNA product flanked by XbaI and XhoI restriction sites was cloned into the corresponding restriction sites of pET21d vector for the expression of C-terminally 6xHis tagged enzyme.
A second round of PCR using the megaprimer and a primer beyond the opposite restriction site was used to generate a product flanked by both restriction sites and carrying the desired mutation.
A second round of PCR using the megaprimer and a primer beyond the second att site was used to generate a product flanked by both att sites and carrying the desired mutation.
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