Circular DNA

Introduction

In this page you will be able to find information about how to convert plasmids received from approved DNA providers to be compatible with the eProtein Discovery™ system using the Circular eGene™ Prep Kit NC3015.
You can consult our previous knowledge base page 01 - Technology section at the voice Circular eGene™ Constructs (Link) where you will find all the information about our product and how to order them.

Circular eGene Prep Content

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	18 µL	Green	-80°C	EBV3-01
Expression Enhancer Buffer	35 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

Table 1. List of components in the eGene Prep kit - Solubility Tag Screen - NC3015.

User supplied reagents / components (not included in the kit)

Circular eGene™ constructs 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

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™ construct

Important Note

Clean your bench and pipettes with 70% ethanol or isopropanol before starting the work.
We recommend using nuclease-free filter tips throughout the protocol to prevent cross-contamination.

Thaw the Expression Enhancer Buffer on ice.
Thaw the Circular eGene™ constructs 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 eGene™ constructs. 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™ construct 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™ construct to the transfer plate
The remaining sample can be sealed and stored in -80 °C.

Nuclera Technical Support:
UK Phone +44 1223 942 761
US Phone: +1 508-306-1297
Email: techsupport@nuclera.com

Offices:
Nuclera UK (HQ):
One Vision Park, Station Road, Cambridge, CB24 9NP, UK

Nuclera USA: 1000 Technology Park Drive, Suite B, Billerica MA 01821, USA www.nuclera.com

Introduction​

Circular eGene Prep Content​

User supplied reagents / components (not included in the kit)​

User supplied equipment​

eGene preparation workflow​

Preparation of circular eGene™ construct​

Preparation of Enhancer Mix​

Preparation of circular-eGene:​

Dilution of circular-eGene:​

Transfer and Storage of circular-eGene​