6/25/2023 0 Comments Transcribe template![]() In the process of PCR reaction, the concentrations of primers should be adjusted and tested according to the difference in the length of primers. However, it is noteworthy that the design and concentrations of primers in the whole experiment are the key factors affecting the quality of products. We demonstrated that the DNA templates of sgRNA synthesized were practicable by using this method. 3a–c show the representative sequences of the transcription template of EGFR-Exon19, pu57-1, pu57-2. coli colonies to confirmed by Sanger’s sequencing. In order to further verified the sequence of the templates, the PCR products were subcloned into PMD18-T vector and we chose three E. In Additional file 1: Table S3, the final combined fragment labeled by AF1-AF2-AF3-Tracr-R, the longest products, was the template of sgRNA and the size of expected DNA fragments similarly assembled by this means were 122 bp. The size of DNA template fragments of the three genes were around 120 bp. The sgRNA DNA template synthesized by overlapping primers was shown on electrophoresis photograph (Fig. However, conventional methods of generating sgRNA either direct synthesis sgRNA or requires plasmid construction for PCR amplification to make transcription templates, which is either expensive or cumbersome and requires several steps to produces gRNAs. Distinct from ZFN (zinc finger nucleases) and TALENS (transcription activator-like effectors), CRISPR/Cas9 system identifies the target site using sgRNA and cleaves target sequence to generate DNA double strand breaks (DSBs) with Cas9 nuclease and then initiates different repair mechanisms of non-homologous end joining (NHEJ) or homology-directed repair (HDR) to produce different types of mutation. It is composed of Cas9 nuclease, CRISPRRNA (crRNA) and trans-activating crRNA (tracrRNA), or a single guide RNA (sgRNA) fused with crRNA and tracrRNA. ![]() ![]() Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system is first found in bacterial and archaea, as an immune defense system to protect against the invasion of viruses and plasmids.
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