TrueTag Donor DNA Kit

Create transfection-ready donor DNA for knock-in experiments

Introducing Invitrogen TrueTag Donor DNA Kit, the fastest and easiest way to create high-quality donor DNA to tag your gene. The TrueTag Donor DNA kit is designed to maximize editing efficiency and significantly reduce protocol time compared to competitor products because all cloning steps have been eliminated. TrueTag is the newest addition to our complete cell engineering solutions that are optimized and designed to work together to minimize the trial-and-error phase and help you get answers with less effort. The kit comes complete with all the necessary reagents to prep the donor DNA and easy to follow protocols help ensure that even novice users can perform successful knock-ins in your own lab.

  • Eliminate cloning steps—just add primers and get results in hours, not days
  • Minimize screening time—select only edited cells using blasticidin or puromycin
  • Easily identify edited cells—tag your gene on the N- or C-terminus with either GFP, RFP, or luciferase
  • Up to 100% edited cells—get great results, regardless of your level of expertise

Make the choice to get truly amazing and reliable results faster than ever before—only the best for your research.

N-terminal or C-terminal tagging of ACTB in U2OS cells

Fluorescence images of U2OS cells expressing beta actin-GFP following TrueTag homology directed repair knock in.
Figure 1. TrueTag knock-in to U2OS cells. U2OS cells were transfected with TrueCut Cas9 v2, a TrueTag dsDNA donor for homology directed repair (HDR) to insert GFP at the N-terminal or C-terminal of the ACTB locus, and a TrueGuide gRNA for either the N-terminal or C-terminal of ACTB. Transfection was performed with Lipofectamine CRISPRMAX reagent. 7 days after transfection, the cells were counterstained with NucBlue Live ReadyProbes Reagent and images were captures on an EVOS FL Color Imaging System.

Reduce donor DNA preparation from days to hours and eliminate cloning steps

Schematic diagrams of TrueTag HDR knock in workflow compared to competitor’s workflow
 Click image to enlarge
Schematic diagrams of TrueTag HDR knock in workflow compared to competitor’s workflow
 Click image to enlarge

Figure 2. Comparison of the TrueTag Donor DNA Kit workflow and a competitor’s homologous recombination knock in workflow. The TrueTag workflow can be done in one afternoon and is as simple as completing a single PCR, column purifying the double-stranded DNA product, and then transfecting the donor (with appropriate CRISPR-Cas9 or TAL effector nucleases) to knock-in the tag into your gene of interest. Conversely, competitor kits require multiple primer designs and PCR steps to assemble the donor molecule into a backbone vector, and subsequent subcloning. This requires additional screening of E. coli colonies as well as sequence verification. The entire process takes several days to complete. The TrueTag system allows for donor molecules to be generated in just a few hours.

Generate stable cell lines with >99% editing efficiency after selection

Fluorescence images and flow cytometry scatter plots showing increases in expressing cells with selection from day 1 to day 10.

Figure 3. Stable cell lines generated in 10 days. Day 1 – 293FT cells are transfected with TrueCut Cas9 v2, a TrueTag dsDNA donor for homology directed repair at the C-terminal of the ACTB locus to insert GFP-puromycin, and a TrueGuide gRNA for the C-terminal of ACTB, with the Lipofectamine CRISPRMAX reagent. TrueTag donor generation is quick process that takes 3 hours or less to complete. On Day 3 or 4, homology directed repair is complete, and the fusion tag is expressed off the native loci. Approximately 25% of the cells in this example were GFP positive. Selection can begin 72 to 96 hours after transfection. By Day 10, this population of cells stability selected with puromycin is >99% positive for GFP. Data collected on an Attune NxT Flow Cytometer.

Using GFP and RFP to track more than one protein in the same cell

Fluorescence images of HEK293 cells expressing both HISTH4C-GFP and RFP-ACTB following TrueTag homology directed repair knock in.

Figure 4. Dual tagging HEK293 cells with GFP and RFP. 293FT cells were transfected with TrueCut Cas9 protein v2, a TrueTag dsDNA donor for homology directed repair (HDR) at the C-terminal of the HIST1H4C locus with GFP-puromycin, a second TrueTag dsDNA donor for HDR at the N-terminal of the ACTB locus with blasticidin-RFP, and TrueGuide Synthetic gRNA targeting each genomic loci. Transfection was performed with Lipofectamine CRISPRMAX reagent. Selective pressure was applied to the 293FT cells to generate a stable pool: 5 days of blasticidin selection was performed first, followed by 5 days of puromycin selection. Cells were counterstained NucBlue Live ReadyProbes reagent and images were captures on an EVOS FL Color Imaging System.

User Guide: TrueTag Donor DNA Kit 

CRISPR validated protocols
Getting started with CRISPR? Need proven CRISPR validated protocols? Our CRISPR Validated Protocols collection has been developed by our expert R&D team. We have matched each of the validated and optimized protocols with the best products to help reduce trial and error and to accelerate your rate of discovery. These step-by-step CRISPR protocols have been optimized for maximum efficiency, viability, and reproducibility across a broad range of cell types and gene targets. We truly want you to succeed with your CRISPR experiment the first time and have confidence in your data.

Access the latest set of protocols

Related products

N-terminal or C-terminal tagging of ACTB in U2OS cells

Fluorescence images of U2OS cells expressing beta actin-GFP following TrueTag homology directed repair knock in.
Figure 1. TrueTag knock-in to U2OS cells. U2OS cells were transfected with TrueCut Cas9 v2, a TrueTag dsDNA donor for homology directed repair (HDR) to insert GFP at the N-terminal or C-terminal of the ACTB locus, and a TrueGuide gRNA for either the N-terminal or C-terminal of ACTB. Transfection was performed with Lipofectamine CRISPRMAX reagent. 7 days after transfection, the cells were counterstained with NucBlue Live ReadyProbes Reagent and images were captures on an EVOS FL Color Imaging System.

Reduce donor DNA preparation from days to hours and eliminate cloning steps

Schematic diagrams of TrueTag HDR knock in workflow compared to competitor’s workflow
 Click image to enlarge
Schematic diagrams of TrueTag HDR knock in workflow compared to competitor’s workflow
 Click image to enlarge

Figure 2. Comparison of the TrueTag Donor DNA Kit workflow and a competitor’s homologous recombination knock in workflow. The TrueTag workflow can be done in one afternoon and is as simple as completing a single PCR, column purifying the double-stranded DNA product, and then transfecting the donor (with appropriate CRISPR-Cas9 or TAL effector nucleases) to knock-in the tag into your gene of interest. Conversely, competitor kits require multiple primer designs and PCR steps to assemble the donor molecule into a backbone vector, and subsequent subcloning. This requires additional screening of E. coli colonies as well as sequence verification. The entire process takes several days to complete. The TrueTag system allows for donor molecules to be generated in just a few hours.

Generate stable cell lines with >99% editing efficiency after selection

Fluorescence images and flow cytometry scatter plots showing increases in expressing cells with selection from day 1 to day 10.

Figure 3. Stable cell lines generated in 10 days. Day 1 – 293FT cells are transfected with TrueCut Cas9 v2, a TrueTag dsDNA donor for homology directed repair at the C-terminal of the ACTB locus to insert GFP-puromycin, and a TrueGuide gRNA for the C-terminal of ACTB, with the Lipofectamine CRISPRMAX reagent. TrueTag donor generation is quick process that takes 3 hours or less to complete. On Day 3 or 4, homology directed repair is complete, and the fusion tag is expressed off the native loci. Approximately 25% of the cells in this example were GFP positive. Selection can begin 72 to 96 hours after transfection. By Day 10, this population of cells stability selected with puromycin is >99% positive for GFP. Data collected on an Attune NxT Flow Cytometer.

Using GFP and RFP to track more than one protein in the same cell

Fluorescence images of HEK293 cells expressing both HISTH4C-GFP and RFP-ACTB following TrueTag homology directed repair knock in.

Figure 4. Dual tagging HEK293 cells with GFP and RFP. 293FT cells were transfected with TrueCut Cas9 protein v2, a TrueTag dsDNA donor for homology directed repair (HDR) at the C-terminal of the HIST1H4C locus with GFP-puromycin, a second TrueTag dsDNA donor for HDR at the N-terminal of the ACTB locus with blasticidin-RFP, and TrueGuide Synthetic gRNA targeting each genomic loci. Transfection was performed with Lipofectamine CRISPRMAX reagent. Selective pressure was applied to the 293FT cells to generate a stable pool: 5 days of blasticidin selection was performed first, followed by 5 days of puromycin selection. Cells were counterstained NucBlue Live ReadyProbes reagent and images were captures on an EVOS FL Color Imaging System.

User Guide: TrueTag Donor DNA Kit 

CRISPR validated protocols
Getting started with CRISPR? Need proven CRISPR validated protocols? Our CRISPR Validated Protocols collection has been developed by our expert R&D team. We have matched each of the validated and optimized protocols with the best products to help reduce trial and error and to accelerate your rate of discovery. These step-by-step CRISPR protocols have been optimized for maximum efficiency, viability, and reproducibility across a broad range of cell types and gene targets. We truly want you to succeed with your CRISPR experiment the first time and have confidence in your data.

Access the latest set of protocols

Related products

TrueTag Donor DNA Kit FAQs

Do I have to use your Cas9 enzyme?
No, our donor DNA kit can be used with any genome editing tool that causes a double stranded DNA break but, for best results, our TrueCut Cas9 protein v2 has the performance on the market.

Does the TrueTag system only induce gene knockouts?
The TrueTag system is a not a gene trapping kit. Our C-terminal donor DNA knock-ins will not disrupt the coding of your gene and will minimize changes in protein function. These donor kits are an easy way to tag your protein and visualize its localization in living cells without deleting the gene.

Do I need to be an expert in gene editing experiments?
Generating your donor DNA construct requires very little expertise in molecular biology techniques. The minimal homology arms mean you only have to look up ~70 bases of DNA, and our dedicated support team can help design PCR oligos if you get stuck.

Will I be able to visualize my protein?
Yes, the TrueTag system is available with three different fluorescent markers (GFP, RFP, and luciferase) that work with the most common filter sets of microscopes that utilize fluorescent detection. This also allows for simultaneous tagging and visualization of different proteins.

Why would I use the TrueTag system versus dye- and antibody-based methods for analyzing my protein?
Sometimes it is difficult to find a dye or an antibody that is specific to your protein of interest. The TrueTag system also eliminates some of the difficulties associated with staining protocols that have to be repeated each experiment.

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Resources

  • Learning Center
    Access genome editing application resources for more success as you plan and execute your experiments.
  • CRISPR Validated Protocols
    Getting started with CRISPR? Need proven CRISPR validated protocols? Check out our collection of step-by-step validated protocols.
  • FAQs
    Find answers to everyday problems, we have consolidated a list of most commonly asked questions.
  • Webinar Series
    Master the art of CRISPR editing by tuning into our on-demand 3 part webinar series.
  • CRISPR Hands-on Workshop
    Our experienced team has designed a comprehensive four-day workshop to get you up to speed on CRISPR.

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