The QIAprep& CRISPR Kit offers a streamlined workflow, which combines a liquid-based sample preparation step that can be completed in only 25 minutes with sensitive PCR and Sanger sequencing detection (see figure The QIAprep& CRISPR Kit workflow).
Cell processing and sample preparation
Cultured CRISPR-edited cells are briefly washed to remove cell culture medium, extracellular material released by living cells and intracellular material released by any dead, lysed cells. Removal of this material is recommended, since it can interfere with downstream sample preparation and amplification processes. For the preparation of CRISPR-edited genomic DNA, the Cell Lysis Buffer supplemented with Proteinase K is directly added to the cells.
The Cell Lysis Buffer included in the QIAprep& CRISPR Kit is optimized for efficient cell lysis, increased lysate stability and high compatibility with the AllTaq PCR chemistry. The lysis reaction can accommodate a broad range of cell numbers and takes place either in a tube or in the culture plate at room temperature. The lysis is stopped at 80°C, and the raw cell lysate can be directly used as input DNA for the PCR reaction with no intermediary purification steps.
Assays for amplifying genomic regions of interest
CRISPR-Q Custom PCR Assays can be easily designed and ordered for human, mouse or rat gene targets using the intuitive custom builder tool available in GeneGlobe at www.geneglobe.com/customize/crispr/ (see figure CRISPR assay design is optimized for human, mouse and rat gene targets). The custom builder tool generates several target-specific assays based on the genomic location and the sequence of the guide RNA (gRNA) used for your particular CRISPR gene editing events.
PCR amplification of genomic regions of interest
PCR amplification of the genomic region of interest can be used to assess the success of the CRISPR gene editing event in the cultured cells. In addition to efficient cell lysis, the QIAprep& CRISPR Kit also provides a highly compatible PCR chemistry: the AllTaq PCR chemistry allows reliable amplification of target genes from raw cell lysates produced with the Cell Lysis Buffer and CRISPR-Q Custom PCR Assays or other PCR primers.
AllTaq Master Mix
The AllTaq Master Mix provides a convenient format for highly sensitive and specific hot-start PCR using any DNA template. The ready-to-use Master Mix contains the AllTaq DNA Polymerase, AllTaq PCR Buffer and dNTPS and is provided as a 4x concentrate, which allows for a higher sample input.
Q-Solution is an additive that facilitates amplification of difficult templates by modifying the melting behavior of nucleic acids. Q-Solution often enables or improves suboptimal PCR caused by DNA templates that have a high degree of secondary structure or that are GC-rich.
Master Mix Tracer
The Master Mix Tracer is an orange dye that enables visual tracking during PCR setup and serves as a loading dye for agarose gels. The dye runs at approximately 50 bp on a 1% agarose gel. The 125x concentrate can either be added to the PCR reaction mix or directly to the master mix stock vial to obtain a 1x final concentration.
CRISPR-Q Control PCR Assay
The CRISPR-Q Control PCR Assay is provided as a 20x concentrate and can be used as a positive control in the PCR (see figure The QIAprep& CRISPR Kit includes a PCR control to help determine lysate quality). It amplifies a conserved target region in human, mouse and rat and alerts you to issues such as insufficient DNA input or insufficient lysate quality. The size of the control PCR product is 261 bp.
Primers for Sanger sequencing of genomic regions of interest
CRISPR-Q Sanger Primers can be easily designed and ordered using the intuitive custom builder tool available in GeneGlobe at www.geneglobe.com/customize/crispr/. This tool generates several target-specific primers based on the genomic location and the sequence of the (gRNA) used for your particular CRISPR gene editing events (see figure The CRISPR-Q Sanger Primers successfully sequence target regions of interest).