1. PolyJet DNA In Vitro Transfection Reagent ----- A Protocol for Transfections of
Mammalian Cell l
500 l l
15875 Gaither Drive
Gaithersburg, MD 20877
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Cat # SL Store at 4 0C
This product is for laboratory research ONLY and not for diagnostic use
Introduction:
Based on our innovative polymer synthesis technology,
PolyJet™ DNA In Vitro Tranfection Reagent is formulated to be
a powerful transfection Reagent that ensures effective and
reproducible transfection with less cytotoxicity. PolyJet™ was
shown to deliver genes to various established cell lines as well
as primary cells.
Procedures for Transfecting Adherent Mammalian
Cells:
Cell Seeding (see Table 1):
Cells should be plated 18 to 24 hours prior to transfection so
that the monolayer cell density reaches to the optimal
80~90% confluency at the time of transfection. Serum-free
DMEM medium or serum-containing medium (less than 5%
serum, e.g., Opti™-Medium from Invitrogen®) without
antibiotics is changed to replace complete serum-containing
culture medium 30 minutes before transfection.
Note: High serum levels (5~10%) usually do not have
inhibitory effect on PolyJet™-mediated transfections.
For some cells, maximal transfection efficiencies are
observed in the absence of serum. So we strongly
suggest conducting transfection in absence of serum as a starting point.
Table 1. A Guideline for Seeding Adherent Cells Prior to
Transfection in Different Culture Formats
Preparation of PolyJet™-DNA Complex and
Transfection Procedures
For different cell types, the optimal ratio of PolyJet™
(L):DNA (g) varies from 2:1 to 3:1. We recommend
the PolyJet™ (L):DNA (g) ratio of 3:1 as a starting
point which usually gives satisfactory transfection
efficiency with invisible cytotoxicity. To ensure the
optimal size of PolyJet™/DNA complex particles, we
recommend using serum-free DMEM with High Glucose
to dilute DNA and PolyJet™ Reagent.
The following protocol is given for transfection in 24-well
plates, refer to Table 2 for transfection in other culture
formats. The optimal transfection conditions for a majority
of adherent cell lines, as well as a general starting point for
optimization are given in the standard protocol described
below.
- For each well, dilute 1.0 µg of DNA into 50 µl of serum-free
DMEM with High Glucose. Vortex gently and spin down
briefly to bring drops to the bottom of the tube.
- For each well, dilute 3.0 µl of PolyJet™ reagent into 50 µl
of serum-free DMEM with High Glucose. Vortex gently and
spin down briefly bring drops to the bottom of the tube.
- Add the diluted PolyJet™ solution to the diluted DNA
solution all at once. (Important: do not
mix the solutions in the reverse order !)
- Vortex- mix the solution immediately and spin down
- Incubate for 15~20 minutes at room temperature.
- Add the 100 µl PolyJet™/ DNA mixture drop-wise
onto the medium in each well and homogenize the
mixture by gently swirling the plate.
- For maximal transfection efficiency, change
the medium to complete serum containing medium
4~5 hours post addition of PolyJet™/DNA complex.
- Check transfection efficiency 24 to 48 hours post
transfection.
Table 2. Recommended Amounts for Different Culture
Vessel Formats
Culture Dishes
Surface Area (cm2)
Number of Cells to Seed
100 mm Dish 58
2.2 – 4.4 x 106
60 mm Dish 21
0.9 – 1.8 x 106
35 mm Dish
9.6
3.5 – 7.0 x 105
6-well Plate
4.0 – 8.0 x 105
12-well Plate
3.5
1.5 – 3.0 x 105
24-well Plate
1.9
0.8 – 1.6 x 105
48-well Plate
1.0
4.0 – 8.0 x 104
96-well Plate
0.3
1.2 – 2.4 x 104
Culture Dish
Culture Volume (ml)
Plasmid DNA (g)
Diluent Volume (mL)
PolyJet™ Reagent (L)
48 well plate
0.38
0.5
2 x 0.02 1
6-well plate
1.6 2
2 x 0.1 6
35 mm dish
60 mm dish
4.5 5
2 x 0.25 15
10 cm dish 8
7 - 8
2 x 0.5
T75 flask
2 x 0.75
250 ml flask 50
2 x 1.25
 2007 SignaGen Laboratories
Storage: PolyJet™ DAN In Vitro Transfection Reagent is stable for up to 18 months at +4 0C. This item shipped at ambient temperature