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  • ‹ Back to Gene editing of immune cells
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genome editing protocol Protocol: gene editing of CD34+ cells
Home › Learning centers › Gene function › Gene editing › Gene editing of immune cells › Gene editing of CD3+ Pan T cells

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    • Shasta Single Cell System introduction
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    • SmartChip Real-Time PCR System introduction
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        • Pathogen detection in human samples and food
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        • Antibiotic resistance genes
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        • Genotyping using animal and blood samples
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        • Webinar: Monitoring ARGs in environmental samples
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    • ICELL8 introduction
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      • ICELL8 system vs plate-seq
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        • Webinar: Leveraging single-cell transcriptomics and epigenomics for biomarker discovery
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        • Single-Cell Workshop at 2020 NextGen Omics Series UK
        • The power of full-length scRNA-seq
        • Sign up: cardiomyocyte webinar
      • Technical notes
        • Enhancing biomarker discovery with SMART-Seq Pro kit and ICELL8 cx system
        • ICELL8 cx system target enrichment for fusions
        • ICELL8 cx system reagent formulation and dispense guidelines
        • Improved detection of gene fusions, SNPs, and alternative splicing
        • Full-length transcriptome analysis
        • High-throughput single-cell ATAC-seq
        • Protocol: High-throughput single-cell ATAC-Seq
        • Single-cell identification with CellSelect Software
        • Single-cell analysis elucidates cardiomyocyte differentiation from iPSCs
        • Combined TCR profiling and 5’ DE in single cells
        • Automated, high-throughput TCR profiling
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        • Isolate cells of any size
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    • Apollo library prep system introduction
      • Automated VeriSeq library preparation for PGS
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      • In-tip bead separation on the Apollo system
  • Next-generation sequencing
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        • SMART technology
        • Single-cell mRNA-seq
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        • Enabling long-read RNA sequencing from low-input samples
        • Singular for low input total RNA seq
        • All-in-one cDNA synthesis and library prep from single cells
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        • All-in-one cDNA synthesis and library prep from ultra-low RNA inputs
        • 3' mRNA libraries from single cells (SMART-Seq v4 3' DE Kit)
        • Full-length mRNA-seq for target capture
        • Stranded libraries from single cells
        • Stranded libraries from picogram-input total RNA (v3)
        • Stranded libraries from 100 pg-100 ng total RNA
        • Stranded libraries from 100 ng - 1 ug total RNA
        • Stranded libraries from FFPE inputs (v2)
        • Nonstranded libraries from FFPE inputs
        • Singular and Takara Bio library prep
        • Full-length, single-cell, and ultra-low-input RNA-seq with UMIs
      • Webinars
        • Pushing the limits of sensitivity for single-cell applications
        • Capturing biological complexity by high-resolution single-cell genomics
        • Taking single-cell RNA-seq by STORM
        • STORM-seq Q&A
        • Neural multiomics Q&A
        • Liver metabolic function, dissecting one cell at a time
        • Pushing the limits Q&A
        • Total RNA sequencing of liquid biopsies
        • Liver metabolic function Q&A
        • Automating full-length single-cell RNA-seq libraries
        • Single-cell whole transcriptome analysis
        • Sensitivity and scale for neuron multiomics
      • RNA-seq tips
      • RNA-seq FAQs
    • Technical notes
      • DNA-seq
        • Next-gen WGA method for CNV and SNV detection from single cells
        • Low-input whole-exome sequencing
        • DNA-seq from FFPE samples
        • Low cell number ChIP-seq using ThruPLEX DNA-Seq
        • Detection of low-frequency variants using ThruPLEX Tag-Seq FLEX
        • ThruPLEX FLEX outperforms NEBNext Ultra II
        • Streamlined DNA-seq from challenging samples
        • High-resolution CNV detection using PicoPLEX Gold DNA-Seq
        • ThruPLEX FLEX data sheet
        • Low-volume DNA shearing for ThruPLEX library prep
        • NGS library prep with enzymatic fragmentation
        • Comparing ThruPLEX FLEX EF to Kapa and NEBNext
      • Immune Profiling
        • Get started with BCR/TCR profiling
        • Track B-cell changes in your mouse model
        • Efficient and sensitive profiling of human B-cell receptor repertoire
        • TCRv2 kit validated for rhesus macaque samples
        • Improved TCR repertoire profiling from mouse samples (bulk)
        • TCR repertoire profiling from mouse samples (bulk)
        • BCR repertoire profiling from mouse samples (bulk)
        • Improved TCR repertoire profiling from human samples (bulk)
        • TCR repertoire profiling from human samples (single cells)
        • BCR repertoire profiling from human samples (bulk)
      • Epigenetic sequencing
        • ChIP-seq libraries for transcription factor analysis
        • ChIP-seq libraries from ssDNA
        • Full-length small RNA libraries
        • Methylated DNA-seq with MBD2
      • Reproductive health technologies
        • Embgenix GT-omics for TE biopsies
        • Embgenix ESM Screen
        • Embgenix PGT-A
    • Technology and application overviews
      • Embgenix GT-omics Oncology Tech Note
      • Sequencing depth for ThruPLEX Tag-seq
      • Whole genome amplification from single cells
    • FAQs and tips
      • Positive and negative controls in scRNA-seq
      • DNA-seq FAQs
      • ChIP-seq FAQs
      • Indexing FAQs
      • TCR-seq methods: Q&A
    • DNA-seq protocols
      • Using UMIs with ThruPLEX Tag-Seq FLEX
      • Targeted capture with Agilent SureSelectQXT
      • Exome capture with Illumina Nextera Rapid Capture
      • Targeted capture with Roche NimbleGen SeqCap EZ
      • Targeted capture with IDT xGen panels
      • Targeted capture with Agilent SureSelectXT
      • Targeted capture with Agilent SureSelectXT2
    • Bioinformatics resources
      • Cogent NGS Analysis Pipeline
        • Cogent NGS Analysis Pipeline notices
      • Cogent NGS Discovery Software
        • Cogent NGS Discovery Software notices
      • Cogent NGS Immune Profiler
        • Cogent NGS Immune Profiler Software notices
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      • Embgenix Analysis Software
      • SMART-Seq DE3 Demultiplexer
    • Webinars
      • Harnessing the power of full-length transcriptome analysis for biomarker discoveries
      • SMART-Seq Pro kits for biomarker detection
      • Takara Bio Single-Cell Workshop, Spring 2021
      • Single-Cell Workshop at 2020 NextGen Omics Series UK
      • Immunogenomics to accelerate immunotherapy
      • MeD-Seq, a novel method to detect DNA methylation
      • Single-cell DNA-seq
    • Posters
      • Long-read mRNA-seq poster
  • Spatial biology
    • Trekker FAQs
  • mRNA and cDNA synthesis
    • mRNA synthesis
      • mRNA synthesis selection guide
      • mRNA synthesis FAQs
      • Takara IVTpro mRNA Synthesis System
        • Cloning Kit for mRNA Template
        • Takara IVTpro T7 mRNA Synthesis Kit
      • 5-prime capping of mRNA
        • Post-transcriptional capping
        • Co-transcriptional capping
      • Download resources
        • Sign up to download our infographic
      • Webinar: Streamlining your IVT workflow to maximize mRNA yields
    • cDNA synthesis
      • Premium total and poly A+ RNA
      • SMARTer RACE 5'/3' Kit
      • Cloning antibody variable regions
  • Gene function
    • Gene editing
      • Gene editing product finder
      • Gene editing tools and information
        • sgRNA design tools
        • Tools for successful CRISPR/Cas9 genome editing
        • Gene editing posters
        • Customer data for Guide-it products
        • How to design sgRNA sequences
        • Introduction to the CRISPR/Cas9 system
        • Gene editing of CD3+ T cells and CD34+ HSCs
      • CRISPR/Cas9 knockouts
        • Mutation detection kit comparison
        • Screening for effective guide RNAs
        • Monoallelic versus biallelic mutants
        • Indel identification kit for mutation characterization
      • CRISPR/Cas9 knockins
        • Choosing an HDR template format
        • Homology-directed repair FAQs
        • Mouse CRISPR knockin protocol
        • Site-specific gene knockins using long ssDNA
        • Efficient CRISPR/Cas9-mediated knockins in iPS cells
        • Oligo design tool for detecting precise insertions
          • Oligo design tool user guide (insertions)
      • Genome-wide screening
        • CRISPR library screening
        • CRISPR library screening webinar
        • Phenotypic screen using sgRNA library system
      • Creating and screening for SNPs
        • SNP detection with knockin screening kit
        • Oligo design tool for SNP screening
          • Oligo design tool user guide (SNPs)
        • Sign up: SNP engineering webinar
        • Guide-it SNP Screening Kit FAQs
      • CRISPR/Cas9 delivery methods
        • Electroporation-grade Cas9 for editing in diverse cell types
        • CRISPR/Cas9 gene editing with AAV
        • CRISPR/Cas9 gesicles overview
        • Cas9 Gesicles—reduced off-target effects
        • sgRNA-Cas9 delivery to many cell types
        • Tet-inducible Cas9 for gene editing
      • Cre recombinase
        • Control your Cre recombinase experiments
        • Fast Cre delivery with gesicle technology
    • Viral transduction
      • Recombinant virus comparison
      • Product finder
      • Transduction posters
      • Lentivirus
        • Lenti-X Transduction Sponge overview
          • Lenti-X Transduction Sponge tech note
          • Lenti-X Transduction Sponge compatible cell types
        • Webinars
          • Webinar: Cellular reprogramming of cancer cells for immunotherapy
          • Lentiviral particles webinar
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        • Lenti-X GoStix Plus video protocols
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        • Rapid lentivirus titration by p24 ELISA
        • Lentiviral particles
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    • T-cell transduction and culture
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      • Hematopoietic cell transduction
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        • Reprogramming PBMCs
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      • Gene editing in hiPS cells
        • Tagging an endogenous gene with AcGFP1 in hiPS cells
        • Tagging an endogenous gene with a myc tag in hiPS cells
        • Generating clonal hiPS cell lines deficient in CD81
        • Introducing a tyrosinemia-related SNP in hiPS cells
        • Inserting an expression cassette into the AAVS1 locus in hiPS cells
        • Editing hiPS cells using electroporation
        • Editing hiPS cells using gesicle technology
        • Single-cell cloning of hiPS cells
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      • Mir-X microRNA quantification
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genome editing protocol Protocol: gene editing of CD34+ cells
User-generated protocol

Gene editing of CD3+ Pan T cells

Overview Materials Protocol Test case

Overview  

overview

Materials  

ProductCompanyCatalog number
Editing reagents
Guide-it sgRNA In Vitro Transcription Kit Takara Bio 632635
Guide-it sgRNA Screening Kit Takara Bio 632639
Guide-it Recombinant Cas9 (Electroporation-Ready) Takara Bio 632641
Guide-it Long ssDNA Production System Takara Bio 632644
Neon Transfection System Thermo Fisher Scientific MPK5000
Neon Transfection System 10 µL Kit Thermo Fisher Scientific MPK1096
Cell culture
Peripheral blood, CD3+ Pan T Cells, negatively selected AllCells PB009-1F
Corning Falcon Polystyrene Microplate 6 well Corning 351146
Corning Falcon Polystyrene Microplate 48 well Corning 351178
RPMI media Millipore Sigma R0883-100ML
FBS (Tet System Approved FBS) 50 ml Takara Bio 631107
L-glutamine Millipore Sigma G7513
Immunocult Human CD3/CD28/CD2 T cell activator StemCell 10970

Protocol  

Electroporation of CD3+ Pan T cells with the Neon Transfection System

A. Cell thawing and activation

  1. Warm RPMI medium containing 10% FBS.
  2. Quickly thaw the vial of frozen cells in a 37°C bead bath. Wipe the outside of the vial with 70% ethanol.
  3. Transfer cell suspension to a 50-ml conical tube.
  4. Rinse the cells with 1 ml of RPMI medium (with 10% FBS). Add the medium dropwise to the cells while gently swirling the conical tube.
  5. Slowly add medium dropwise to the cells until the total volume is 5 ml, while gently swirling after each addition of several drops of the medium.
  6. Slowly bring the volume up to 15 ml by adding 1–2 ml aliquots of RPMI medium (with 10% FBS) dropwise, while gently swirling after each addition.
  7. Centrifuge the cell suspension at 200g for 15 min at room temperature.
  8. Carefully remove all but 1 ml of the medium by pipetting so the cell pellet is not disturbed. Gently resuspend the cell pellet in the remaining medium.
  9. Slowly bring the volume up to 15 ml by adding no more than 1–2 ml of medium at once and gently swirling the tube after each addition.
  10. Centrifuge the cell suspension at 200g for 15 min at room temperature.
  11. Carefully remove all but 2 ml of the medium by pipetting. Gently resuspend the cell pellet in the remaining 2 ml of medium and count the cells by your preferred method.
  12. Culture the cells at a density of 1 x 106 cells/ml in a humidified incubator at 37°C with 5% CO2 for 24 hr.
  13. After the 24-hr incubation, activate the CD3+ T cells by adding 25 µl of ImmunoCult Human CD3/CD28/CD2 T Cell Activator per ml of cell suspension (following manufacturer's recommendation).
  14. Incubate cells at 37°C and 5% CO2 for up to 48–72 hrs before editing.

B. Preparation of cells and cell-culture medium

  1. After a minimum of 48 hrs of activation, prepare a sufficient quantity of fresh cells for your experiment.

    NOTE: Each electroporation requires 2 x 105 cells. However, due to the potential variation of the pipette and tip volumes, we recommend preparing 1.5X the necessary volume of cell suspension (i.e., 3 x 105 cells) for electroporation with a 10-µl Neon Tip to ensure that there is sufficient volume. In other words, prepare a 15-µl volume of cells, RNP complex and HDR template for use with a 10-µl tip.

  2. Take an aliquot of the activated CD3+ T cell suspension and measure the cell density using your preferred method.
  3. Harvest the cells by centrifugation at 200g for 15 min in a 15-ml conical tube at 4°C. You can keep the supernatant medium for further use to recover the cells after electroporation (Section D.11).
  4. Wash the cells once with PBS (without Ca2+ and Mg2+) and then resuspend CD3+ T cells in Resuspension Buffer T (included with Neon kits) at a final concentration of 4 x 104 cells/µl (i.e., 3 x 105 cells in 7.5 µl total volume).
  5. Keep the cell suspension on ice until use.

    NOTE: Minimize the time that T cells are resuspended in Resuspension Buffer T.

  6. Prepare enough fresh medium for culturing the cells after electroporation. You will need 0.125 ml of RPMI medium with 10% FBS for each electroporation reaction.

  7. Aliquot 0.125 ml of the medium prepared in Step 6 into each well of a 48-well plate. Incubate at 37°C and 5% CO2.

C. Preparation of Cas9-sgRNA RNP complex

  1. Thaw Guide-it Recombinant Cas9 (Electroporation-Ready) (Cat. # 632641) and sgRNA solutions at room temperature. In our test case (described below), the sgRNAs were prepared using the Guide-it In Vitro Transcription Kit (Cat. # 632635) and their activities were tested in vitro using the Guide-it sgRNA Screening Kit (Cat. # 632639). Please note that Cat. #s 632635 and 632639 are provided together as the Guide-it Complete sgRNA Screening System (Cat. # 632636).

    NOTE: To avoid repeated freeze/thaw cycles, prepare aliquots upon initial thawing of Guide-it Recombinant Cas9 (Electroporation-Ready).

  2. Combine the following components in a 200-µl PCR tube to mix the Cas9 protein and sgRNA at a 5:1 mass ratio. The molar ratio of Cas9 protein to sgRNA will be approximately 1:1 in this mixture and the total volume will be 7.5 µl. Be sure to use the same buffer that was used to resuspend the cells (Resuspension Buffer T).

    NOTE: The reaction volume indicated below is 1.5X the required volume.

    Per reaction:

    sgRNA (e.g., 700 ng/µl) (or 10.5 pmols) 0.49 μl*
    Guide-it Recombinant Cas9 (3 µg/µl) (or 10.5 pmols) 0.56 μl
    Resuspension Buffer T 6.45 μl*
    Total volume 7.5 μl*

    *The added volume of sgRNA will vary depending on sgRNA concentration, and the added volume of Resuspension Buffer T should be adjusted such that the total reaction volume is 7.5 µl. The volumes indicated above are based on a sgRNA concentration of 700 ng/µl.

    NOTES:

    • If you are performing multiple electroporations, make a master mix and aliquot 7.5 µl per reaction into each PCR tube.

    • To maximize electroporation efficiency, the combined volume of the Cas9 and sgRNA solutions should be ≤20% of the total volume of the Cas9-sgRNA RNP complex reaction mix (e.g., for the 7.5-µl reaction specified above, the combined volume of the sgRNA and Cas9 solutions should be ≤1.5 µl).

    • For knockin experiments, the HDR donor template should be added just before mixing the Cas9-sgRNA RNP complexes with the cell suspension (Section D.3). Adjust the volume of Resuspension Buffer T included in the reaction such that the final volume upon addition of donor DNA is 7.5 µl. To generate the application data presented below, donor DNA was added in varying amounts between 0.5–1 μg.

  3. Mix the reaction well by gently pipetting up and down. Incubate using a thermal cycler preheated to 37°C with the following program:

    37°C 5 min
    4°C Hold

D. Electroporation

  1. Fill the Neon Tube with 3 ml of Buffer E (included with Neon kits) and insert the Neon Tube into the Neon Pipette Station.
  2. Using the touchscreen on the Neon system, set up the electroporation parameters as follows:

    Pulse voltage = 1,600 V
    Pulse width = 10 ms
    Pulse number = 3 pulses
  3. If performing a knockin experiment, add the HDR template to the Cas9-sgRNA RNP complex (Step C.3).
  4. Gently resuspend the cells by tapping and transfer 7.5 µl of the cell suspension into the PCR tube containing the 7.5 µl of Cas9-sgRNA RNP complex solution.
  5. Mix well by gently pipetting up and down.
  6. Insert the Neon Pipette into the Neon Tip and confirm that the pipette and tip are tightly connected.
  7. Using the Neon Pipette, aspirate the mixture of RNP and cells slowly into the Neon Tip.

    NOTE: Avoid any air bubbles in the tip. If you notice air bubbles, place the sample back into the PCR tube and aspirate again into the tip without any bubbles. The presence of air bubbles causes arcing during electroporation, leading to lowered or failed transfection.

  8. Insert the Neon Pipette into the Neon Tube placed in the Neon Pipette Station and run the program detailed in Step 2, above.
  9. Remove the pipette very carefully and transfer the cells into the plate prepared in Section B.7.
  10. Shake the plate appropriately to disperse the cells and incubate in a humidified incubator at 37°C with 5% CO2.
  11. Two hours after electroporation, add 0.125 ml/well of the supernatant medium obtained in Section B.3.

    NOTE: IL-2 and other activators can be used in fresh media instead (Oh, Seki & Rutz, 2019).

  12. Incubate at 37°C and 5% CO2.
  13. Perform analysis of knockout or knockin efficiency 48 hr after electroporation using your desired method.

Test case  

The fusion of AcGFP to endogenous genes TUBA1A and SEC61B

As a test case, we edited CD3+ T cells in two independent experiments to fuse a fluorescent protein (AcGFP1) to two endogenous genes: TUBA1A or SEC61B. The HDR editing efficiency was determined by flow cytometry using the percentage of positive AcGFP cells four days after electroporation.

panel A

Panel B

Sequence 5'→3'
sgRNA TUBA1A GATGCACTCACGCTGCGGGAAGG
sgRNA SEC61B GCCATACCATATTGGAGATGAGG

panel C

Stock concentration Volume
Cas9 protein 3 mg/ml 0.56 µl
sgRNA 700 ng/µl 0.49 µl
HDR template 1 µg/µl X µl
Buffer T N/A 7.5–X µl
CD3+ T cells 4 x 104 cells/µl 7.5 µl

Figure 1. Details of the HDR editing experiment for the fusion of AcGFP1 with two endogenous genes, TUBA1A and SEC61B, in CD3+ T cells. Panel A. Scheme of the overall editing experiment. CD3+ T cells were electroporated with RNP complex together with an HDR template to introduce a specific sequence (encoding for fluorescent protein AcGFP1) in the desired target site (TUBA1A or SEC61B). Panel B. Sequences of the sgRNAs used in this study (PAM sequences depicted in bold). Panel C. Amounts of editing reagents and CD3+ T cells used for both electroporation reactions.


Figure 2. Optimization of HDR editing experiment and flow-cytometry analysis of edited cell population. Panel A. Percentages of AcGFP+ cells obtained in the different editing experiments designed to fuse AcGFP with TUBA1A and SEC61B genes. Different types of HDR templates (single-stranded or double-stranded DNA) as well as different amounts (from 0.5 μg to 1 μg) were tested with both genes. Panel B. (Left) Flow cytometry analysis of AcGFP1 knockin efficiencies at TUBA1A and SEC61B loci in CD3+ T cells. Following electroporation and culturing, the bulk-edited cell population and the nonelectroporated negative control cells were analyzed for AcGFP1 expression by flow cytometry. Knockin efficiencies were determined by measuring the percentage of fluorescent cells in each population (orange gates in the flow cytometry plots). (Right) Microscopy of edited cells. In each case, AcGFP1 localized in a manner consistent with the function of the endogenous protein: microtubules (TUBA1A locus) or endosomal compartments (SEC61B locus), respectively.

References

Oh, S.A., Seki, A., & Rutz, S. (2019). Ribonucleoprotein transfection for CRISRP/Cas9-mediated gene knock out in primary T cells. Curr. Protoc. Immunol., 124, e69. Doi:10.1002/cpim.69.

Related Products

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License Statement

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M54 This product is covered by the claims of U.S. Patent Nos. 7,704,713 and its foreign counterparts. 
272 This product (“Product”) and its use, is the subject of U.S. Patents 8,697,359 and 8,771,945 and pending U.S. Patent applications. The purchase of the Product conveys to the buyer the non-transferable right to use Product(s) purchased from Takara Bio USA, Inc. or its Affiliates, and any progeny, modification or derivative of a Product, or any cell or animal made or modified through use of a Product, or any progeny, modification or derivative of such cell or animal (“Related Material”), solely for research conducted by the buyer in accordance with all of the following requirements. No right is given to use this Product or Related Material for any other purpose, including, but not limited to, use in drugs, in vitro diagnostic purposes, therapeutics, or in humans. (i) The buyer shall not sell or otherwise transfer Products (including without limitation any material that contains a Licensed Product in whole or part) or any Related Material to any other person or entity, or use Products or any Related Material to perform services for the benefit of any other person or entity, (ii) the buyer shall use only the purchased amount of the Products and components of the Products, and shall use any Related Material, only for its internal research and not for (a) the practice, performance or provision of any method, process or service, or (b) the manufacture, sale, use, distribution, disposition or importing of any product, in each case (a) or (b) for consideration, or on any other commercial basis (“Commercial Purpose”), (iii) the buyer shall use Licensed Products and any Related Material in compliance with all applicable laws and regulations, including without limitation applicable human health and animal welfare laws and regulations, and (iv) the buyer shall indemnify, defend and hold harmless MIT, Harvard and The Broad and their current and former trustees, directors, officers, faculty, affiliated investigators, students, employees, and agents and their respective successors, heirs and assigns (“Indemnitees”), against any liability, damage, loss, or expense (including without limitation reasonable attorneys’ fees and expenses) incurred by or imposed upon any of the Indemnitees in connection with any claims, suits, investigations, actions, demands or judgments arising out of or related to the exercise of any rights granted to the buyer, or any breach of the rights granted hereunder by the buyer.
391 LIMITED USE LABEL LICENSE: RESEARCH USE ONLY Notice to Purchaser: This product is the subject to a license granted to Takara Bio USA, Inc. and its Affiliates from Caribou Biosciences, Inc., and this product is transferred to the end-user purchaser (“Purchaser”) subject to a “Limited Use Label License” conveying to the Purchaser a limited, non-transferable right to use the product, solely as provided to Purchaser, together with (i) progeny or derivatives of the product generated by the Purchaser (including but not limited to cells), and (ii) biological material extracted or derived from the product or its corresponding progeny or derivatives (including but not limited to cells) (collectively, the product, and (i) and (ii) are referred to as (“Material”) only to perform internal research for the sole benefit of the Purchaser. The Purchaser cannot sell or otherwise transfer Material to a third party or otherwise use the Material for any Excluded Use. “Excluded Use” means any and all: (a) commercial activity including, but not limited to, any use in manufacturing (including but not limited to cell line development for purposes of bioproduction), product testing, or quality control; (b) preclinical or clinical testing or other activity directed toward the submission of data to the U.S. Food and Drug Administration, or any other regulatory agency in any country or jurisdiction where the active agent in such studies comprises the Material; (c) use to provide a service, information, or data to a third party; (d) use for human or animal therapeutic, diagnostic, or prophylactic purposes or as a product for therapeutics, diagnostics, or prophylaxis; (e) activity in an agricultural field trial or any activity directed toward the submission of data to the U.S. Department of Agriculture or any other agriculture regulatory agency; (f) high throughput screening drug discovery purposes (i.e., the screening of more than 10,000 experiments per day) as well as scale-up production activities for commercialization; (g) modification of human germline, including editing of human embryo genomes (with the sole exception of editing human embryonic stem (ES) cell lines for research purposes) or reproductive cells; (h) self-editing; and/or (i) stimulation of biased inheritance of a particular gene or trait or set of genes or traits (“gene drive”). It is the Purchaser’s responsibility to use the Material in accordance with all applicable laws and regulations. For information on obtaining additional rights, including commercial rights, please contact licensing@cariboubio.com or Caribou Biosciences, Inc., 2929 7th Street, Suite 105, Berkeley, CA 94710 USA, Attn: Licensing.
396 Sigma-Aldrich CRISPR Use License Agreement This Product and its use are the subject of one or more of the following issued patents and patent applications: Australia Patent Nos. 2013355214; 2017204031; and 2018229489; Canada Patent Nos. 2,891,347 and 2,977,152; China Patent No. CN105142669; European Patent Nos. EP 2 928 496 B1; EP 3 138 910 B1, 3 138 911 B1, EP 3 138 912 B1, EP 3 360 964 B1, EP 3 363 902 B1; Israel Patent No. IL238856; Singapore Patent No. 11201503824S; South Korea Patent Nos. 10-1844123 and 10-2006880; and U.S. Patent Application Serial Nos. 15/188,911; 15/188,924; 15/188,927; 15/188,931; and 15/456,204 (the “Patent Rights”). The purchase of this Product conveys to you (the “Buyer”) the NON-TRANSFERABLE right to use the Product for Licensed Research Use (see definition below) subject to the conditions set out in this License Agreement. 1. “Licensed Research Use” means any use for research purposes, except: (i) Buyer may not sell or otherwise transfer the Product (including without limitation any material that contains the Product in whole or part) or any Related Material to any other third party (except that you may transfer the Product, or any Related Material to a bona fide collaborator or contract research organization), or use the Products or any Related Material to perform services for the benefit of any other third party; (ii) Buyer may use only the purchased amount of the Product and components of the Product, and shall use any Related Material, only for your internal research within the Field, and not for any Commercial Purposes; (iii) Buyer shall use the Product and any Related Material in compliance with all applicable laws and regulations, including without limitation applicable human health and animal welfare laws and regulations; and (iv) the Buyer shall indemnify, defend, and hold harmless SIGMA and their current and former directors, officers, employees and agents, and their respective successors, heirs and assigns (the “Indemnities”) against any liability, damage, loss, or expense (including without limitation reasonable attorneys’ fees and expenses) incurred by or imposed upon any of the Indemnitees in connection with any claims, suits, investigations, actions, demands or judgments arising out of or related to the exercise of any rights granted to the Buyer hereunder or any breach of this License Agreement by such Buyer. 2. For purposes of Section 1 above, the following definitions shall apply: “Commercial Purposes” means (a) the practice, performance or provision of any method, process or service, or (b) the manufacture, sale, use, distribution, disposition or importing of any product, in each case (a) or (b) for consideration, or on any other commercial basis. “Field” means use as a research tool for research purposes; provided, however, that notwithstanding the foregoing, the Field shall expressly exclude (a) any in vivo and ex vivo human or clinical use, including, without limitation, any administration into humans or any diagnostic or prognostic use, (b) the creation of transgenic rodent models and/or derivatives thereof (including, but not limited to, rodents’ cells and rodents’ organs) by for-profit entities, (c) any in vivo veterinary or livestock use, or non-research agricultural use, or (d) use as a testing service, therapeutic or diagnostic for humans or animals. “Related Materials” means any progeny, modification or derivative of a Product. 3. Your right to use the Product will terminate immediately if you fail to comply with these terms and conditions. You shall, upon such termination of your rights, destroy all Product, Related Materials, and components thereof in your control, and notify SIGMA of such in writing. For information on purchasing a license to this Product for purposes other than Licensed Research Use, contact your local SIGMA sales representative, or call +1 800-325-3010.

The Guide-it In Vitro Transcription Kit contains the necessary reagents for the transcription and cleanup of your sgRNA sequences. It is combined with the Guide-it sgRNA Screening Kit as part of the Guide-it Complete sgRNA screening system which can be used for the simple production, cleanup, and evaluation of single guide RNAs (sgRNAs) for CRISPR/Cas9 studies.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Production of sgRNA by in vitro transcription and testing for cleavage using recombinant Cas9 protein

Production of sgRNA by in vitro transcription and testing for cleavage using recombinant Cas9 protein
Schematic highlighting the key steps for using Guide-it kits for the production of high amounts of single guide RNA (sgRNA) and screening for target site cleavage efficacy using recombinant Cas9 protein.

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sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System

sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System
sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System. Panel A. 20 µl of each sgRNA in vitro transcription (IVT) reaction was incubated for 4 hr at 37°C. The yield for each transcribed sgRNA was quantified using a NanoDrop spectrophotometer. Panel B. Each sgRNA IVT reaction was a run on an Agilent Bioanalyzer to assay for sample quality.

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sgRNA quality produced via in vitro transcription

sgRNA quality produced via in vitro transcription
sgRNA quality produced via in vitro transcription. sgRNAs produced using the Guide-it sgRNA In Vitro Transcription Kit were compared with sgRNAs produced using a competitor’s kit. The Guide-it kit produced a high-quality, single band for each reaction, while the competitor’s kit showed unwanted byproducts.

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Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs

Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs
Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs.
632639 Guide-it™ sgRNA Screening Kit 50 Rxns USD $475.00

License Statement

ID Number  
391 LIMITED USE LABEL LICENSE: RESEARCH USE ONLY Notice to Purchaser: This product is the subject to a license granted to Takara Bio USA, Inc. and its Affiliates from Caribou Biosciences, Inc., and this product is transferred to the end-user purchaser (“Purchaser”) subject to a “Limited Use Label License” conveying to the Purchaser a limited, non-transferable right to use the product, solely as provided to Purchaser, together with (i) progeny or derivatives of the product generated by the Purchaser (including but not limited to cells), and (ii) biological material extracted or derived from the product or its corresponding progeny or derivatives (including but not limited to cells) (collectively, the product, and (i) and (ii) are referred to as (“Material”) only to perform internal research for the sole benefit of the Purchaser. The Purchaser cannot sell or otherwise transfer Material to a third party or otherwise use the Material for any Excluded Use. “Excluded Use” means any and all: (a) commercial activity including, but not limited to, any use in manufacturing (including but not limited to cell line development for purposes of bioproduction), product testing, or quality control; (b) preclinical or clinical testing or other activity directed toward the submission of data to the U.S. Food and Drug Administration, or any other regulatory agency in any country or jurisdiction where the active agent in such studies comprises the Material; (c) use to provide a service, information, or data to a third party; (d) use for human or animal therapeutic, diagnostic, or prophylactic purposes or as a product for therapeutics, diagnostics, or prophylaxis; (e) activity in an agricultural field trial or any activity directed toward the submission of data to the U.S. Department of Agriculture or any other agriculture regulatory agency; (f) high throughput screening drug discovery purposes (i.e., the screening of more than 10,000 experiments per day) as well as scale-up production activities for commercialization; (g) modification of human germline, including editing of human embryo genomes (with the sole exception of editing human embryonic stem (ES) cell lines for research purposes) or reproductive cells; (h) self-editing; and/or (i) stimulation of biased inheritance of a particular gene or trait or set of genes or traits (“gene drive”). It is the Purchaser’s responsibility to use the Material in accordance with all applicable laws and regulations. For information on obtaining additional rights, including commercial rights, please contact licensing@cariboubio.com or Caribou Biosciences, Inc., 2929 7th Street, Suite 105, Berkeley, CA 94710 USA, Attn: Licensing.

The Guide-it sgRNA Screening Kit contains PCR reagents to amplify genomic DNA from your target cells and recombinant Cas9 for the in vitro screening of your sgRNA to help you pick the best guide sequences before moving to experiments in living cells or animals. It is combined with the Guide-it In VItro Transcription kit as part of the Guide-it Complete sgRNA Screening System.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

Documents Components Image Data

Back

Production of sgRNA by in vitro transcription and testing for cleavage using recombinant Cas9 protein

Production of sgRNA by in vitro transcription and testing for cleavage using recombinant Cas9 protein
Schematic highlighting the key steps for using Guide-it kits for the production of high amounts of single guide RNA (sgRNA) and screening for target site cleavage efficacy using recombinant Cas9 protein.

Back

sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System

sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System
sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System. Panel A. 20 µl of each sgRNA in vitro transcription (IVT) reaction was incubated for 4 hr at 37°C. The yield for each transcribed sgRNA was quantified using a NanoDrop spectrophotometer. Panel B. Each sgRNA IVT reaction was a run on an Agilent Bioanalyzer to assay for sample quality.

Back

sgRNA quality produced via in vitro transcription

sgRNA quality produced via in vitro transcription
sgRNA quality produced via in vitro transcription. sgRNAs produced using the Guide-it sgRNA In Vitro Transcription Kit were compared with sgRNAs produced using a competitor’s kit. The Guide-it kit produced a high-quality, single band for each reaction, while the competitor’s kit showed unwanted byproducts.

Back

Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs

Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs
Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs.

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632639: Guide-it sgRNA Screening Kit

632639: Guide-it sgRNA Screening Kit
631107 Tet System Approved FBS 50 mL USD $120.00

Fetal Bovine Serum (FBS) that has been functionally tested for optimal use with all of our Tet Systems. This FBS does not contain trace levels of tetracycline (or its derivatives) which have been observed to interfere with proper regulation of TRE-controlled gene expression. Use of this serum ensures the maximal range of induction possible with the Tet Systems.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Fetal bovine serum (FBS) approved for use with tetracycline-inducible expression systems

Fetal bovine serum (FBS) approved for use with tetracycline-inducible expression systems

Fetal bovine serum (FBS) approved for use with tetracycline inducible expression systems. Only Takara Bio USA functionally tests FBS to be sure that no contaminating tetracycline derivatives interfere with induced expression levels.

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Our fetal bovine serum is functionally tested to guarantee consistent results with tetracycline-inducible expression systems

Our fetal bovine serum is functionally tested to guarantee consistent results with tetracycline-inducible expression systems

Fetal bovine serum from Clontech is functionally tested to guarantee consistent results with tetracycline-inducible expression systems. Serum from other vendors that have not been functionally tested may contain tetracyclines that affect the maximum expression in Tet-Off systems, or generate background in Tet-On systems. Data here shows induction of luciferase expression from our CHO-AA8-Luc Tet-Off Control Cell Line using different sources of FBS.

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631107: Tet System Approved FBS

631107: Tet System Approved FBS


User-generated protocols

User-generated protocols

User-generated protocols are based on internal proof-of-concept experiments, customer collaborations, and published literature. In some cases, relevant results are discussed in our research news BioView blog articles. While we expect these protocols to be successful in your hands, they may not be fully reviewed or optimized. We encourage you to contact us or refer to the published literature for more information about these user-generated and -reported protocols. 

If you are looking for a product-specific, fully optimized User Manual or Protocol-At-A-Glance, please visit the product's product page, open the item's product details row in the price table, and click Documents. More detailed instructions for locating documents are available on our website FAQs page.

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Takara Bio USA, Inc. provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. As a member of the Takara Bio Group, Takara Bio USA is part of a company that holds a leadership position in the global market and is committed to improving the human condition through biotechnology. Our mission is to develop high-quality innovative tools and services to accelerate discovery.

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Takara Bio USA, Inc. provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. As a member of the Takara Bio Group, Takara Bio USA is part of a company that holds a leadership position in the global market and is committed to improving the human condition through biotechnology. Our mission is to develop high-quality innovative tools and services to accelerate discovery.

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES (EXCEPT AS SPECIFICALLY NOTED).

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  • Great value master mixes
  • Signature enzymes
  • High-throughput real-time PCR solutions
  • Detection assays
  • References, standards, and buffers
  • Stem cell research
  • Media, differentiation kits, and matrices
  • Stem cells and stem cell-derived cells
  • mRNA and cDNA synthesis
  • In vitro transcription
  • cDNA synthesis kits
  • Reverse transcriptases
  • RACE kits
  • Purified cDNA & genomic DNA
  • Purified total RNA and mRNA
  • PCR
  • Most popular polymerases
  • High-yield PCR
  • High-fidelity PCR
  • GC rich PCR
  • PCR master mixes
  • Cloning
  • In-Fusion seamless cloning
  • Competent cells
  • Ligation kits
  • Restriction enzymes
  • Nucleic acid purification
  • Automated platforms
  • Plasmid purification kits
  • Genomic DNA purification kits
  • DNA cleanup kits
  • RNA purification kits
  • Gene function
  • Gene editing
  • Viral transduction
  • Fluorescent proteins
  • T-cell transduction and culture
  • Tet-inducible expression systems
  • Transfection reagents
  • Cell biology assays
  • Protein research
  • Purification products
  • Two-hybrid and one-hybrid systems
  • Mass spectrometry reagents
  • Antibodies and ELISA
  • Primary antibodies and ELISAs by research area
  • Fluorescent protein antibodies
  • Special offers
  • In-Fusion Snap Assembly EcoDry promo
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  • OEM
  • Instrument services
  • Gene and cell therapy manufacturing
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  • Facilities
  • Request samples
  • FAQs
  • Instrument services
  • Apollo services
  • ICELL8 services
  • SmartChip ND system services
  • Gene and cell therapy manufacturing
  • Services
  • Facilities
  • Our process
  • Resources
  • Sales
  • Make an appointment with your sales rep
  • Online tools
  • GoStix Plus FAQs
  • Vector information
  • Vector document overview
  • Vector document finder
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  • Automation systems
  • Next-generation sequencing
  • Spatial biology
  • Real-time PCR
  • Nucleic acid purification
  • mRNA and cDNA synthesis
  • PCR
  • Cloning
  • Stem cell research
  • Gene function
  • Protein research
  • Antibodies and ELISA
  • Automation systems
  • Shasta Single Cell System introduction
  • SmartChip Real-Time PCR System introduction
  • ICELL8 introduction
  • Next-generation sequencing
  • RNA-seq
  • Technical notes
  • Technology and application overviews
  • FAQs and tips
  • DNA-seq protocols
  • Bioinformatics resources
  • Webinars
  • Spatial biology
  • Trekker FAQs
  • Real-time PCR
  • Download qPCR resources
  • Overview
  • Reaction size guidelines
  • Guest webinar: extraction-free SARS-CoV-2 detection
  • Technical notes
  • Nucleic acid purification
  • Nucleic acid extraction webinars
  • Product demonstration videos
  • Product finder
  • Plasmid kit selection guide
  • RNA purification kit finder
  • mRNA and cDNA synthesis
  • mRNA synthesis
  • cDNA synthesis
  • PCR
  • Citations
  • PCR selection guide
  • Technical notes
  • FAQ
  • Cloning
  • Automated In-Fusion Cloning
  • In-Fusion Cloning general information
  • Primer design and other tools
  • In‑Fusion Cloning tips and FAQs
  • Applications and technical notes
  • Stem cell research
  • Overview
  • Protocols
  • Technical notes
  • Gene function
  • Gene editing
  • Viral transduction
  • T-cell transduction and culture
  • Inducible systems
  • Cell biology assays
  • Protein research
  • Capturem technology
  • Antibody immunoprecipitation
  • His-tag purification
  • Other tag purification
  • Expression systems
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  • Interview: adapting to change with Takara Bio
  • Applications
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  • Partnering
  • Contact us
  • mRNA and protein therapeutics
  • Characterizing the viral genome and host response
  • Identifying and cloning protein targets
  • Expressing and purifying protein targets
  • Immunizing mice and optimizing vaccines
  • Pathogen detection
  • Sample prep
  • Detection methods
  • Identification and characterization
  • SARS-CoV-2
  • Antibiotic-resistant bacteria
  • Food crop pathogens
  • Waterborne disease outbreaks
  • Viral-induced cancer
  • Immunotherapy research
  • T-cell therapy
  • Antibody therapeutics
  • T-cell receptor profiling
  • TBI initiatives in cancer therapy
  • Cancer research
  • Kickstart your cancer research with long-read sequencing
  • Sample prep from FFPE tissue
  • Sample prep from plasma
  • Cancer biomarker quantification
  • Single cancer cell analysis
  • Cancer transcriptome analysis
  • Cancer genomics and epigenomics
  • HLA typing in cancer
  • Gene editing for cancer therapy/drug discovery
  • Alzheimer's disease research
  • Antibody engineering
  • Sample prep from FFPE tissue
  • Single-cell sequencing
  • Reproductive health technologies
  • Embgenix FAQs
  • Preimplantation genetic testing
  • ESM partnership program
  • ESM Collection Kit forms
  • Infectious diseases
  • Develop vaccines for HIV
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  • Career spotlights
  • Current events
  • Customer stories
  • Gene editing
  • Research news
  • Single-cell analysis
  • Stem cell research
  • Tips and troubleshooting
  • Women in STEM
  • That's Good Support!
  • About our blog
  • That's Good Science!
  • SMART-Seq Pro Biomarker Discovery Contest
  • DNA extraction educational activity
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