Circular eGene™ Preparation Kit
Circular eGene™ Prep Kit
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The Circular eGene™ Prep Kit NC3015 is desgined to convert plasmids received from approved DNA provider to be compatible with the eProtein Discovery™ system.
When placing an order for the Circular eGene™ (plasmids), complete the ordering form with your DNA sequence of choice and select vectors based on the solubility tag options available. Solubility tag, detector tag and Strep purification tag is encoded within the vector sequences, adding these elements to the N- and C-terminus of your protein-of-interest. The schematic of the plasmid structure can be visualised in Figure 1.
Contents
The Circular eGene Prep kit - NC3015 comes with the components listed in Table 1.
The components are supplied in a Nuclera branded box with a green stripe on the label, and must be used within 18 months from the manufacturing date indicated on the kit box.
| Component | Volume | Cap Color | Storage Temperature | Tube reference number |
|---|---|---|---|---|
| Expression Enahncer | 180 µL | Green | -80°C | EBV3-01 |
| Expression Enhancer Buffer | 350 µL | Yellow | -80°C | EBV3-02 |
| Circular eGene Dilution Reagent | 5 mL | White | -80°C | EBV3-03 |
| Nuclease-free Water (NFW) | 100 µL | Blue | -80°C | EBV3-04 |
User supplied reagents / components (not included in the kit)
▷ Circular eGenes at a concentration of 100 nM
▷ Nuclease-free filter tips
▷ 96-well PCR plate or 200 µl PCR strip or Nuclease free 1.5 mL microcentrifuge tubes
User supplied equipment
▷ 37°C incubator or thermocycler
▷ P2, P10, P100 and P200 pipettes
eGene preparation workflow
Figure 2. Circular eGene workflow summary - timings applicable for generating circular eGene constructs using the Circular eGene Prep Kit. For each step of the workflow, the clock indicates the total experiment time and the hand indicates the total hands-on time.
Preparation of circular eGene
Clean your bench and pipettes with 70% ethanol or isopropanol before starting the work.
We recommend using nuclease-free fitler tips throughout the protocol to prevent cross-contamination.
▷ Thaw the Expression Enhancer Buffer on ice.
▷ Thaw the Circular eGenes on ice.
Preparation of Enhancer Mix
▷ Label 1.5 mL microcentrifuge tubes as "Enhancer Mix."
▷ Once the Expression Enhancer solutions are completely thawed, briefly spin them to collect all liquid at the bottom of the tube.
▷ Using a 20 µL pipette, add 14 µL of Expression Enhancer, 14 µL nuclease-free water into the pre-labelled microcentrifuge tube. Using a 100 µl pipette add 28 µL of Expression Enhancer Buffer to the same tube.
▷ Set the pipette volume to 50 µL and gently mix by pipetting up and down 12 times. Avoid generating bubbles.
▷ Spin down the Enhancer Mix to collect the liquid at the bottom of the tube.
Preparation of circular-eGene:
▷ Label 24 microcentrifuge tubes or use a PCR plate for each of the 24 circular eGenes. If using a PCR plate, ensure you have a clear and accurate map indicating the position of each gene.
▷ Add 2 µL of the Enhancer Mix to each well/tube using a 2 or 10 µl pipette.
▷ Spin down the plates/tubes at 1000 rpm for 30 sec.
▷ Using a 10 μl pipette, Pipette 8 μL of 100 nM Circular eGene received from your DNA vendor of choice into each labeled tube or PCR plate well (one eGene per tube/well)
▷ Using a 10 µL pipette, gently mix by pipetting up and down 10 times.
▷ If using a PCR plate, seal it with adhesive. Make sure that sealing is good and does not lead to evaporation of the reaction mix.
▷ Spin down the tubes/PCR plate at 1000 rpm for 30 sec.
▷ Incubate at 37 °C for 30 min in a thermocycler or incubator.
▷ If using a thermocycler, set the lid temperature to 47 °C.
Dilution of circular-eGene:
▷ After incubation, remove the tubes/plate from the thermocycler/incubator and briefly spin them.
▷ To each tube or well, add 150 µL of Circular eGene Dilution Reagent.
▷ Gently pipette to mix the solution thoroughly.
▷ Briefly centrifuge the tubes/PCR plate at 1000 rpm for 30 sec.
Transfer and Storage of circular-eGene
▷ Transfer 5 µL of the diluted circular eGene to the Transfer Plate
▷ The remaining sample can be sealed and stored in -80 °C.