This product is a kit for obtaining sgRNA by in vitro transcription. It consists of PCR amplification reagent (for amplifying dsDNA template for in vitro transcription) and in vitro transcription reagent. Using the backbone sequence specifically recognized by Cas9 as a template, a specially designed upstream specific primer (containing the T7 promoter sequence, the target gene specific sequence, and the overlapping sequence of the backbone portion) is used to generate dsDNA under the action of DNA polymerase, and then 20 μg of functional sgRNA is synthesized from this dsDNA by using the dsDNA as a template (the amplified template can be used for transcription directly without purification) through the T7 RNA polymerase. g of functional sgRNA can be synthesized by T7 RNA polymerase.
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a genomic locus found in many bacteria and archaea, and the CRISPR/Cas9 system is an adaptive immune defense system developed by bacteria and archaea over a long period of time to combat invading viruses and exogenous DNA. It has been found that CRISPR/Cas9 can be used as a gene editing technology to specifically cut DNA target sites in cells, which may result in gene knockout or knock-in during cellular repair of damage, and ultimately achieve the purpose of genomic DNA modification.
Cas9 (CRISPR-associated protein 9) is a double-stranded DNA endonuclease; sgRNA (Single Guide RNA) consists of 2 parts, a single-stranded RNA consisting of a 20-nucleotide crRNA (CRISPR RNA) sequence that binds to a specific DNA target, and a transactivating crRNA (transactivating crRNA) sequence that binds to the Cas9 protein. Single-stranded RNA consisting of a 20-nucleotide crRNA (CRISPR RNA) sequence that binds to a specific DNA target and a tracrRNA (transactivating crRNA) sequence that binds to the Cas9 protein. sgRNAs containing 20 nt of the target sequence bind to Cas9 proteins that have double-stranded DNA endonuclease activity to form a reactive nuclear ribonucleoprotein (RNP) that will specifically cleave the target DNA duplex, resulting in a double-stranded break in the DNA duplex.
| individual parts making up a compound | norm |
| 2X ApexHF FS PCR Master Mix | 625 μl X 2 pcs |
| Scaffold Template ( 2 ng / μl ) *1 | 250 μl |
| Control sgRNA Oligo (10 μM)*2 | 5 μl |
| In Vitro Transcription Buffer | 875 μl |
| RNase Inhibitor ( 40 U/μl ) | 62.5 μl |
| T7 RNA polymerase (100 U/μl ) | 125 μl |
| DNase I ( RNase free ) (5 U/μl ) | 100 μl |
| 2X RNA Loading Dye | 250 μl |
| RNase free water | 1 ml X 2 pcs |
*2: Control sgRNA Oligo is the control sgRNA upstream primer.
Storage temperature: -20℃ storage
Transportation temperature: -20℃ ice bag transportation or dry ice transportation
