Our sgRNA Design Center designed 8 sgRNAs per gene for multiple genes in mice and conducted
in vitro transcription within 2 days, producing 10-20 μg sgRNAs for rapid use in cytological experiments.
Experimental Procedures:
One-step synthesis of gRNA DNA template
Experimental Results:
1. The gRNA DNA template was cloned into the pUC57 vector using blunt-end ligated and verified with Sanger sequencing. The comparison results were consistent with the designed sequence. As shown in Figure 1.
Figure 1. Plot of blunt-end ligated results against the design sequence
2. Electrophoresis in 2% agarose was performed on the sgRNA obtained by in vitro transcription and clear bands could be seen. This is shown in Figure 2.
Figure 2. sgRNA agarose gel electropherogram
3.One sgRNA sequence was verified: The selected sgRNA was reverse transcribed to obtain cDNA, sgRNA amplification primers were designed, and the DNA sequence of sgRNA was obtained through PCR. Then the DNA sequence was cloned into the pUC57 vector using blunt-end ligation and verified with Sanger sequencing. The resulting sequencing data verified that the sgRNA sequence was correct. This is shown in Figure 3.
Figure 3. sgRNA sequence validation agarose gel electrophoretogram
* Lane1: sgRNA reverse transcribed cDNA; Lane2: DNaseI treated sgRNA; Lane3: DNA template for in vitro transcription
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