Genome editing/ CRISPR-Cas9

Prokaryotic and eukaryotic cells

Genetic engineering terms the targeted modifications of genetic information of living organisms/cells. Genome editing stands for targeted engineering by using specifically designed nucleases. The recent CRISPR/ Cas-9 method uses RNA-guided nuclease. That makes the process faster, easier and more specific compared to other gene editing methods. Since the DNA-binding element is RNA it is simple and cheap to manipulate and it allows sequence specific programming. The high targeting efficiency, results in significant time saving, e.g., for the generation of knockout mice. In basic research CRISPR is mainly used in cell line engineering and animal genetic engineering (e.g. of disease models) to investigate gene function.

Vector generation

Vector generation

Restriction and ligation enzymes are used to generate the plasmid vector. This process requires incubation at specific temperatures, e.g. in the Eppendorf Thermomixer, to ensure optimal reaction conditions. Also the amplification of specific linear DNA fragments by PCR might be necessary.

  • Thermo mixing devices
    • Mixing, heating, and cooling in one instrument for high flexibility
    • All common vessels and plate formats from 5 µL to 50 mL by different Eppendorf SmartBlocks for standard applications
    • Excellent 2DMix-Control technology results in excellent sample mixing
    • Anti-spill technology prevents lid wetting and cross-contamination
    • Increased mixing frequency of up to 3,000 rpm for effective sample mixing
  • PCR consumables
    • One-piece design: combining a polycarbonate frame and polypropylene wells for optimum performance
    • Extremely thin-walled polypropylene wells guarantee optimum heat transfer to the sample
    • Exceptionally solid and torque-resistant polycarbonate frame
    • Improved well-to-well tolerance
    • Cut-away corner and alphanumeric grid-referencing
  • Tubes
    • Hinged Eppendorf Safe-Lock lid prevents unintentional lid opening during incubation and storage
    • Polypropylene provides high resistance to chemicals, mechanical stress and temperature extremes
    • Manufactured without the use of slip agents, plasticizers, and biocides – substances that have been shown to leach from plastic consumables into the sample and negatively affect bio-assay results
    • g-Safe®: Exceptional centrifugation stability up to 30,000 × g prevents sample loss due to tube breakage and provides extra safety when working with hazardous samples
    • Precise lid sealing for minimal evaporation rates during long-term storage
  • Pipettes and tips
    • The unique one-button operation offers a fast and ergonomic handling with reduced operating effort and active aerosol reduction. To avoid an accidential tip ejection a clear haptic feedback is provided.
    • The spring loaded tip cone improves user to user reproducibility and improved ergonomics by lowering tip attachment force
    • The Reference 2 multi channel offers a high flexibility incl a spring-loaded tip cone which can be switched on/off optionally
    • The secondary adjustment supports the easy adjustment of your Reference 2 for the most accurate pipetting of different liquids or other external conditions without the need for a full calibration
    • The quick lock enables an easy cleaning and maintenance of your pipette
  • Thermocyclers
    • Intuitive graphic programming
    • Small footprint
    • Universal block for ultimate flexibility
    • Connect up to 2 Mastercycler nexus eco to your existing unit
    • Available with gradient
Transformation and cultivation (prokaryotic cells)

Transformation and cultivation (prokaryotic cells)

The generated plasmid vector has to be introduced into bacteria by a transformation process. Common methods are electroporation, e.g. with the Eppendorf Eporator or via heat shock. For the heat shock technique the Eppendorf Thermomixer as well as the ThermoStat can be used.

  • Electroporator
    • Faster sample handling – simple one-button operation
    • Maximum safety via integrated cuvette holder
    • Self-explanatory operation guided by display indicators
    • Fast, freely programmable softkeys for saving and accessing the 2 most frequently used parameter settings
    • Compact, space-saving design
  • Centrifuges
    • Max. rotor capacity: 24 × 1.5/2.0 mL vessels
    • Max. speed: 20,238 × g (14,680 rpm)
    • OptiBowl® design for quiet operation, even without a rotor lid
    • Soft-touch one-finger lid closure for ergonomic operation
    • SOFT brake to protect sensitive samples
  • Shaker
    • Stackable – up to three units for maximum space savings
    • Programmable Innova controls automatically changes temperature, speed, and optional photosynthetic and UV germicidal lights at timed intervals
    • Versatile accessory platform (sold separately) accommodates flasks up to 5 L
    • Slide-out platform mechanism – provides easy and effortless access to flasks located in the front and back of the shaker
    • Built-in water reservoir humidifies chamber to reduce sample evaporation, while also protecting unit from spills, includes a drain for easy cleaning
  • Pipettes and tips
    • Automatic Combitip recognition: Eliminates time consuming volume calculations, avoiding incorrect dispensing volumes
    • Ergonomic one-button tip ejector: For one handed operation and contact-free Combitips advanced ejection
    • Speed Adjustment: Program aspiration and dispensing speed for the utmost in precision and accuracy while also preventing splashing
    • Repetitive dispensing modes: Decrease the number of movements for the user by allowing them to fill the tip once and dispense up to 100 times without a refill
    • Wide dispensing range: 1 μL to 50 mL provides up to 5,000 dispensing volumes with increments as low as 100 nanoliters
  • Photometers
    • Absorption measurements for one or more fixed wavelengths
    • Scan for specific UV applications with automatically determined purity ratios
    • Preprogrammed applications for quick start and applications with evaluation via factor, standard or standard series
    • Integrated application and results memory
    • Data transfer via USB interface and direct printout of results
Preparation and purification

Preparation and purification

After transformation, screening for suitable clones takes place. Centrifuges with a kit rotor are advantageous for this step. The concentration and purity of the isolated DNA will be measured in a spectrophotometer. And if sequencing is required, conventional PCR builds up the basic reaction.

  • Thermocyclers
    • Intuitive graphic programming
    • Small footprint
    • Universal block for ultimate flexibility
    • Connect up to 2 Mastercycler nexus eco to your existing unit
    • Available with gradient
  • Thermo mixing devices
    • Mixing, heating, and cooling in one instrument for high flexibility
    • All common vessels and plate formats from 5 µL to 50 mL by different Eppendorf SmartBlocks for standard applications
    • Excellent 2DMix-Control technology results in excellent sample mixing
    • Anti-spill technology prevents lid wetting and cross-contamination
    • Increased mixing frequency of up to 3,000 rpm for effective sample mixing
  • Photometers
    • Absorbance measurement for one or more wavelengths, recording of wavelength scans from 200 nm to 830 nm (increment: 1 nm)
    • Automatic evaluation and storage of measurement data with clear presentation of results
    • Spectral graph to display sample purity with automatic ratio calculation
    • Preprogrammed applications for quick start
    • Freely programmable applications with evaluation via factor, standard or standard series and freely programmable parameters
  • Accessories (e.g. Kit rotor)
  • Concentrator
    • Comes in 3 system configurations
    • Extremely quite operation <50 dB(A)
    • Chemical-resistant, maintenance-free PTFE diaphragm pump eliminates the need for changing pump oil
    • Small footprint saves valuable bench space
    • Wide variety of 15 different rotor options to accommodate tubes from 0.2 mL to 50 mL, microplates and PCR plates
Transfection and cultivation (eukaryotic cells)

Transfection and cultivation (eukaryotic cells)

During transfection the molecules will be delivered into eukaryotic cells to edit their genetic information. This can be done by transfection methods like electroporation or microinjection. After that the eukaryotic cells have to be seeded into cell culture consumables and cultured in a CO2 incubator.

  • CO2 Incubators
    • Temperature range from 4 °C above ambient to 50 °C
    • Shaking speeds between 25 – 400 rpm, 25 – 300 rpm if stacked, provides versatility for culturing a wide range of cell types
    • Large 35.6 × 61 cm (14 in × 24 in) platform accommodates flasks up to 4 liters providing flexibility with your cultures
    • Single or double stacked for increased capacity
    • Inner and outer door saves valuable gasses while maintaining the perfect enviornment for your cells
  • Dishes, flasks and plates
    • Unsurpassed safety in handling during transportation and while working due to corrugated handling ring
    • SplashProtect ring inside of the dish lid traps liquid and prevents spills during transportation or incubation
    • Pronounced rim on dish lid and exact fitting of lid and bottom ensure sturdy, secure stacking
    • Easy differentiation of lid and dish bottom prevents unintended removal of the lid
    • Packaging combines tool-free opening, resealable top and side for tape-free closing, and an easy solution to shrink the package for space-conscious storage
  • Conical tubes
    • Premium USP class 6 raw material and no use of slip agents, plasticizers, and biocides during manufacturing ensure highest sample integrity, high g-Safe® centrifugation stability, and optimal sample and pellet visibility.
    • Cap with flattened and grooved sides supports a user-friendly ergonomic handling, enables a stable upright positioning, and minimizes contamination risk.
    • Reliable and safe labelling due to a large writing area on the tube and due to a flat and light-colored cap.
    • Precise dimensions enable maximum compatibility with centrifuge rotors, mixers and shakers.
    • Sterile, pyrogen-, DNase-, RNase-, and DNA-free. Each individual lot is checked and certified for quality and purity.
  • Micromanipulators
    • Unique DualSpeed™ joystick for precise and instantaneous movement control
    • Maximum stability ensures vibration-free work
    • Angle adjustment from 0° to 90°
    • One-hand operation for capillary and angle settings
    • Programmable Z-axis limit for preventing capillary breakage
  • Microinjectors
    • Maximum stability ensures drift-free work
    • Electronic coupling to FemtoJet microinjectors for fast serial semi-automatic injections with highest reproducibility
    • Selection and programming of advanced functions (e.g. axial movement, step injection)
    • Ergonomically shaped control panel for fatigue-free work
    • Easy and precise angle adjustment
Harvesting and preparation

Harvesting and preparation

After cultivating the manipulated cells, they have to be harvested by centrifugation and counted in order to determine their number. Due to the fact that these cells are very valuable consumables of high purity grades should be chosen for the preparation procedure.

  • Centrifuges
    • Max. rotor capacity: 48 × 1.5/2.0 mL, 6 × 50 mL, 2 × MTP
    • Max. speed: 30,130 × g (17,500 rpm)
    • Remarkable versatility with 12 different rotors
    • Soft-touch one-finger lid closure for ergonomic operation
    • Menu-driven, multi lingual operation menu (English, German, French, Spanish) with large backlit display
  • Conical tubes
    • Premium USP class 6 raw material and no use of slip agents, plasticizers, and biocides during manufacturing ensure highest sample integrity, high g-Safe® centrifugation stability, and optimal sample and pellet visibility.
    • Cap with flattened and grooved sides supports a user-friendly ergonomic handling, enables a stable upright positioning, and minimizes contamination risk.
    • Reliable and safe labelling due to a large writing area on the tube and due to a flat and light-colored cap.
    • Precise dimensions enable maximum compatibility with centrifuge rotors, mixers and shakers.
    • Sterile, pyrogen-, DNase-, RNase-, and DNA-free. Each individual lot is checked and certified for quality and purity.
  • Thermo mixing devices
    • Mixing, heating, and cooling in one instrument for high flexibility
    • All common vessels and plate formats from 5 µL to 50 mL by different Eppendorf SmartBlocks for standard applications
    • Excellent 2DMix-Control technology results in excellent sample mixing
    • Anti-spill technology prevents lid wetting and cross-contamination
    • Increased mixing frequency of up to 3,000 rpm for effective sample mixing
  • Biopur® consumables
    • Hinged Eppendorf Safe-Lock lid prevents unintentional lid opening during incubation and storage
    • Polypropylene provides high resistance to chemicals, mechanical stress and temperature extremes
    • Manufactured without the use of slip agents, plasticizers, and biocides – substances that have been shown to leach from plastic consumables into the sample and negatively affect bio-assay results
    • g-Safe®: Exceptional centrifugation stability up to 30,000 × g prevents sample loss due to tube breakage and provides extra safety when working with hazardous samples
    • Precise lid sealing for minimal evaporation rates during long-term storage
  • Tubes
    • Hinged Eppendorf Safe-Lock lid prevents unintentional lid opening during incubation and storage
    • Polypropylene provides high resistance to chemicals, mechanical stress and temperature extremes
    • Manufactured without the use of slip agents, plasticizers, and biocides – substances that have been shown to leach from plastic consumables into the sample and negatively affect bio-assay results
    • g-Safe®: Exceptional centrifugation stability up to 30,000 × g prevents sample loss due to tube breakage and provides extra safety when working with hazardous samples
    • Precise lid sealing for minimal evaporation rates during long-term storage
Analysis and detection

Analysis and detection

Depending on which analysis methods are suitable for detecting the success of the experiment, one or more molecule types (DNA, RNA or proteins) have to be isolated and analyzed. After quantification and quality control by photometric measurements, further downstream analytic follows.

  • Photometers
    • Absorbance measurement for one or more wavelengths, recording of wavelength scans from 200 nm to 830 nm (increment: 1 nm)
    • Automatic evaluation and storage of measurement data with clear presentation of results
    • Spectral graph to display sample purity with automatic ratio calculation
    • Preprogrammed applications for quick start
    • Freely programmable applications with evaluation via factor, standard or standard series and freely programmable parameters
  • Cuvettes
    • Suitable for measuring small volumes (≥50 µL)
    • UV and Vis measurements from 220 nm to 1,600 nm
    • UV transparent plastic, free of fluoropolymers and other halogenated hydrocarbons
    • Two built-in optical path lengths in a single cuvette - just turn the UVette 90° to change from 10 mm and 2 mm
    • Volume markings for 500 µL and 1,000 µL
  • LoBind consumables
    • LoBind material guarantees maximum sample recovery for improved assay results
    • Free of surface coating (e.g., silicone) to minimize the risk of sample interference
    • Lot-certified PCR clean purity grade: of human DNA, DNAase, RNase and PCR inhibitors
    • Available in tube, microplate, and deepwell plate formats for easy-up scaling
    • Precise lid sealing to minimize evaporation
  • Thermocyclers
    • Intuitive graphic programming
    • Small footprint
    • Universal block for ultimate flexibility
    • Connect up to 2 Mastercycler nexus eco to your existing unit
    • Available with gradient
  • PCR consumables
    • One-piece design: combining a polycarbonate frame and polypropylene wells for optimum performance
    • Extremely thin-walled polypropylene wells guarantee optimum heat transfer to the sample
    • Exceptionally solid and torque-resistant polycarbonate frame
    • Improved well-to-well tolerance
    • Cut-away corner and alphanumeric grid-referencing