Precision gRNA Synthesis Kit - FAQs

Low RNA yield can be due to several factors, including the pH of the binding buffer. Another common issue is RNA degradation. We recommend taking all precautions to prevent RNase activity. Some customers have reported an increase in yield by modifying the ethanol concentrations in the purification steps:
- 60% ethanol in the binding step (Step 2)
- 67% ethanol in Wash Buffer 1
- 80% ethanol in Wash Buffer 2
These modifications are detailed in the CellEvent Senescence Green Detection Kit (Pub. No. MAN0018221 A.0) .

If you continue to experience issues, please contact technical support at technicalsupport@thermofisher.com.

Find additional tips, troubleshooting help, and resources within our PCR and cDNA Synthesis Support Center.

The only complete way to confirm that there are no off-target effects is to sequence the entire genome of your cell. Alternatively, a less thorough means of checking for off-target editing is to perform targeted sequencing of sequences with the highest probability of off-target effects (i.e., most similar to your CRISPR target region).

A single guide RNA (gRNA) is all that is required for targeting, but we do recommend testing 2-3 gRNAs against each locus being targeted for cleavage. Testing multiple gRNAs increases the chances of finding a gRNA with high editing efficiency, which will reduce the screening time required to identify the clone of interest.

The Lipofectamine CRISPRMAX Reagent combined with the proprietary enhancement properties of the Lipofectamine Cas9 Plus Reagent leads to efficient complex formation with the Cas9-gRNA ribonucleoprotein, for best delivery to the nucleus, helping to ensure high gene editing frequency for a wide range of cell types.

Although the Lipofectamine CRISPRMAX Reagent was developed for the delivery of the Cas9-gRNA ribonucleoprotein, there is potential for other protein complex applications.

The Lipofectamine CRISPRMAX Reagent was developed to efficiently deliver the Cas9-gRNA ribonucleoprotein complex. We recommend that you first deliver the donor plasmid with Lipofectamine 3000, then follow with delivering the Cas9-gRNA complex with Lipofectamine CRISPRMAX Reagent for best editing efficiency.

The Precision gRNA Synthesis Kit (Cat. No. A29377) includes primers for synthesis of gRNA targeting safe harbor locus HPRT. We also have a fully processed, fully validated HPRT gRNA with GCD primers for confirmation of cleavage available from our custom services team.

No other kits are required for this process. All necessary components are included in the kit.