BTN Protocol Guide-08

��| Gene ��Transfer
��Transfection
Roc
��
he
��
Applied Science
��
9115 Hague Road,
��
Indianapolis,
��
IN 46250
Sales:
��
800-262-1640
��
Tec
��
hnical:
��
800-262-4911
��
bioc
��
hemts.us@roc
��
he.com
��
www.roche-applied-science.com/transfection
��
Simple Optimization Protocol for Difﬁ
��
cult to
��
Transfect Cells Using FuGENE
�
�
®

��
HD
��
Transfection Reagent
��
Some cell lines are considered
��
difﬁ cult to transfect because the
��
3. Return the cell plates
��
to the incubator and lea
��
ve
�
the transfection
��
correct parameters for transfection of tha
��
t cell line are not known.
��
complex pla
��
te at room temperature.
��
��
The
��
rea
��
gent:DNA
��
ratio must
��
be tested because laboratories may
��
��
quantify DNA amounts slightly differently. The amount
��
of complex
��
4. Wait 15 minutes,
��
then
��
repeat the complex
��
addition, this time in
��
added is an equally critical factor; too much complex may result the wells on the right side of each cell plate.
��
in cytotoxicity, and too little
��
complex
��
may result in suboptimal
��
��
expression. When testing new cell lines,
��
we use a simple protocol
��
5. Return the cell plates to the incubator until
��
time of assay (24-72
��
which provides comparisons between four critical parameters:
��
hours depending upon gene expressed).
��
• Ratio of µl of reagent to µg of DNA
��
• ��
• Amount of complex added to the cells
��
6. Prior to assaying the cells for expression of the transfected
• ��
• Length of time for complex to form gene, visually inspect the cells. Figure 1 shows the data from an
• ��
• Cell density
��
experiment in which only two amounts of complex
�
were added
• ��
to the plates; the protocol
��
is easily
��
modiﬁ ed
��
to suit individual
Day 1
��
needs.
��
1. Plate the cells in two 96-well plates at two different densities,
��

��
using antibiotic-free complete medium. These are the cell plates.
��
��
Day 2
��
1. Prepare the transfection complex in serum-free medium:
��
�
��
��
��
��
a. Calculate volume needed. Prepare diluted plasmid at 20 µg/
��
��
ml.
��
��
b. Form complexes in an empty 96-well round bottom plate;
��
��
this plate is the transfection complex plate. Complexes are ��
��
formed on this pla
��
te; 2 wells contain controls:
��
��
��
• The reagent control (top) well contains 150 µl
��
serum-free
��
��
medium and 12 µl FuGENE
®
��
HD
��
Transfection Reagent. ��
• The plasmid control (bottom) well contains 150 µl diluted
��
��
� � � � � � � � � � � � � � � � � � � � � � � �
��
plasmid.
��
• ��
• The six comple
��
x wells (middle) each contain 3 µg of diluted
��
plasmid in 150 µl medium and 12, 10.5, 9, 7.5, 6, and 4.5
��
��
�
��
•
µl FuGENE
®
HD Transfection Reagent, respectively. This
��
��
��
produces six different ratios of reagent to DNA.
• ��
��
2. As soon as the last complex
��
has
��
been
��
formed, remove the
��
cell
�
��
pla
�
tes
��
from the incuba
��
tor
��
. Gently add
��
different amounts of
��
complex
��
to the wells
��
on the left side of each cell plate, such
that ﬁ ve different complex amounts are added to each of the six
��
complex ratios, and each row on the pla
��
te corresponds to one of
��
the six different complex
��
ratios from the complex plate. The same
��
plate layout is used for both cell plates.
��
��
��
��
Protocol Guide ı 2008 View entire protocol online at www.biotechniques.com/protocol ı BioTechniques ı 33
ProtocolGuide06-07.indd 27 10/10/06 4:10:14 PM
Roche protocol.indd 33 10/26/07 11:23:20 AM
Page 1 | Page 2 | Page 3 | Page 4 | Page 5 | Page 6 | Page 7 | Page 8 | Page 9 | Page 10 | Page 11 | Page 12 | Page 13 | Page 14 | Page 15 | Page 16 | Page 17 | Page 18 | Page 19 | Page 20 | Page 21 | Page 22 | Page 23 | Page 24 | Page 25 | Page 26 | Page 27 | Page 28 | Page 29 | Page 30 | Page 31 | Page 32 | Page 33 | Page 34 | Page 35 | Page 36 | Page 37 | Page 38 | Page 39 | Page 40 | Page 41 | Page 42 | Page 43 | Page 44 | Page 45 | Page 46 | Page 47 | Page 48 | Page 49 | Page 50 | Page 51 | Page 52 | Page 53 | Page 54 | Page 55 | Page 56 | Page 57 | Page 58 | Page 59 | Page 60 | Page 61 | Page 62 | Page 63 | Page 64 | Page 65 | Page 66 | Page 67 | Page 68 | Page 69 | Page 70 | Page 71 | Page 72 | Page 73 | Page 74 | Page 75 | Page 76