Biology Reference
In-Depth Information
Table 1
Preparation of CAT Reaction Mixture
Number of Samples to be
Solution
15
30
45
60
0.25 M Tris-HCl
0.375 mL
0.75 mL
1.125 mL
1.5 mL
5 mM Chloramphenicol
0.75 mL
1.5 mL
2.25 mL
3.0 mL
H
2
O
1.875 mL
3.75 mL
5.625 mL
7.5 mL
[
3
H] Acetyl CoA
3
µ
L
6
µ
L
9
µ
L
12
µ
L
Total Volume
3 mL
6 mL
9 mL
12 ml
tube, thus the dual luciferase system provides speed and reliability for the assay.
RCS cells, a cell line derived from rat chondrosarcoma, produce a number of
proteins specific to chondrocytes and have been used for study of cartilage
gene regulation
(4)
. Unlike LipofectAMINE, FuGene is a nonliposomal based
reagent and can be used for transfection in the presence of serum. FuGene has
become popular because it produces high levels of transfection of many cell
types with minimum optimization and less cytotoxicity.
1.
Plate RCS cells the d before the transfection to achieve about 1 - 3
×
10
5
cells in
a six-well dish. It is not necessary to wash cells prior to transfection.
2. Prepare FuGene 6 reagent/DNA complex by the manufacturer's protocol
(3-6
g of DNA). DNA solution contains the
two reporter constructs
742Luc/Int
and
pRL-SV40
at 10:1 ratios, 1
µ
L FuGene 6 reagent per 1-2
µ
µ
g DNA
per a six-well plate. (
See
Notes 7
and
8
.)
3.
Dropwise, add mixture of FuGene 6 reagent/DNA to the cells. Swirl the wells to
ensure even dispersal.
4.
After incubation of the cells for 48 h, wash the wells and add 200
L lysis buffer
per well (PLB, Promega Dual-Luciferase Kit). Scrape the cells and transfer the
cell suspension to a 1.5-mL microcentrifuge tube. Lysis the cells by freezing and
thawing in dry ice and 37
µ
°
C water bath twice. Remove debris by centrifuging the
lysate for 2 min.
5.
The cleared lysate (50 mL) is used to measure activity for
firefly
and
Renilla
luciferase sequentially by a luminometer.
3.4. Transgenic Mice
The purity of DNA is important for the successful production of transgenic
mice. Plasmids can be purified by banding on CsCl two times. However, plas-
mid preparation kits from Qiagen also work well if the samples are not over-
loaded in the column purification step. The kits are more convenient than the
CsCl method.
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