Using the Neon
™
Transfection System
Introduction
Instructions are provided in this section to use the Neon
™
device with the
Neon
™
Pipette Station and Neon
™
Kits for electroporation of mammalian cells.
General instructions to prepare cells for use with the Neon
™
Transfection
System are described below. For primary and stem cell types, use the
established methods developed in the laboratory.
See page 22 if you wish to use the preprogrammed optimization protocol.
Materials Needed
Ordering information is on page 38.
Cells
Neon
™
Kits
High quality DNA at a concentration of 1–5 μg/μL in deionized water or
TE buffer, or high quality RNAi duplex at a concentration of 100–250 μM
in nuclease-free water (page 13)
Cell culture plates containing the appropriate medium
D-PBS or Phosphate buffered saline (PBS) without Ca
2+
and Mg
2+
(page 38)
Trypsin/EDTA or TrypLE
™
Express (Cat. no. 12563) for adherent cells
Countess
®
Automated Cell Counter (page 38) or equivalent
R
E
C
O
MM
ENDA
TI
O
N
If you are a first time user of the Neon
™
Transfection System, we recommend
that you review the protocol below and ensure that you are able to insert and
use the Neon
™
Pipette and Tip correctly into the Neon
™
Pipette Station (see
below for details) before you start using the system with your samples.
Important
To obtain the highest transfection efficiency and low non-specific effects,
optimize transfection conditions by varying electrical parameters as
described on page 22 using the pre-programmed optimization protocol in a
24-well format.
Since the cell culture conditions vary from user to user, be sure to use low
passage number, actively dividing cells (for dividing cells)
For siRNA transfection, the concentration of RNAi duplex required will vary
depending on the efficacy of the duplex. After the initial results, vary the
siRNA final concentration from 10–200 nM.
Note:
The siRNA concentration in the Neon
™
transfection protocol refers to
the siRNA concentration in the culture medium and not to the siRNA
concentration in the electroporation mix in the Neon
™
Tip.
Continued on next page
14
