Biology Reference
In-Depth Information
4.
Culture media
. Dulbecco's Modifi ed Eagle's Medium
(DMEM, Gibco
®
, Life Technologies, Carlsbad, CA, USA;
#430027); fetal bovine serum (FBS, Gibco
®
; #10099-141);
Episerf (Gibco
®
; #10732-022); phosphate-buffered saline
(PBS, Gibco
®
; #10010015)
5.
Trypsinization medium
. trypsin-EDTA in PBS 1:250
(0.05 %/0.02 %), Phenol Red, without Ca
++
and Mg
++
,
(BioConcept Ltd, Allschwil, Switzerland; #5-51 F00-I).
Store at 4 °C
6.
Transfection mix
. CaCl
2
0.5 M (73.5 g CaCl
2
· 2H
2
O; com-
plete to 500 ml with H
2
O); HBS (28 ml 5 M NaCl, 11.9 g
HEPES, 750
l 1 M Na
2
HPO
4
in 400 ml H
2
O, complete to
500 ml, pH 7.1) (
see
Note 2
). Aliquot and store at −20 °C
7.
Cell lysis
. Benzonase (Novagen
®
, Merck KGaA, Darmstadt,
Germany; #70664-3); 3-[(3-Cholamidopropyl)
dimethylammonio]-1-propanesulfonate (CHAPS, AppliChem
GmbH, Darmstadt, Germany; #A1099)
8.
Iodixanol gradient
. 30 ml Optiseal tube (Beckman Coulter,
Indianapolis, IN; tube #361625 and spacers #392833 for
70Ti rotor); Optiprep solution (60 % iodixanol; Axis-shield,
Dundee, Scotland); PBS-MK solution is obtained by mixing
500 ml of D-PBS (Gibco
®
; #14040174) with 50 mg
MgCl
2
· 6H
2
O; PBS pH 7.4 (Gibco
®
; #10010056)
9.
Heparin affi nity chromatography
. 1 ml HiTrap Heparin col-
umn (GE Healthcare, Little Chalfont, UK; #17-0406-01);
bidistilled H
2
O (Gibco
®
; #15230089)
10.
Vector concentration and storage
. Amicon Ultra-15 centrifugal
fi lter unit (Millipore, Billerica, MA, USA; #UFC910024, cut-
off 100 kDa); Amicon Ultra-4 centrifugal fi lter unit (Millipore;
#UFC810024, cut-off 100 kDa); D-PBS with Ca
++
and Mg
++
(Gibco
®
; #14040174); Maxymum Recovery 0.6 ml tubes
(Axygen Inc., Union City, CA, USA; #MCT-060-L-C)
μ
1.
Cells
. HEK 293 T/17 (American Type Culture Collection
(ATCC), Manassas, USA)
2.
Isolation of genomic DNA
. NucleoSpin
®
Tissue columns
(Macherey-Nagel, Düren, Germany) (
see
Note 3
); Nuclease
S1 (Promega, Madison, WI, USA; # M5761)
3.
Quantitative PCR
. Primer set for human albumin gene—for-
ward primer:
5
2.2 AAV6 Vector
Titration and Quality
Control
′
TGA AAC ATA CGT TCC CAA AGA GTT
T
3
′
, reverse primer:
5
′
CTC TCC TTC TCA GAA AGT GTG
CAT AT
3
, probe:
FAM-
TGC TGA AAC ATT CAC CTT CCA
TGC AGA
-BHQ-1
; primer set for the pAAV-cmv-GFP vector
(human
′
β
-globin intronic region)—forward primer:
5
′
CGT
GCC AAG AGT GAC GTA AG
3
′
, reverse primer:
5
′
TGG
TGC AAA GAG GCA TGA TA
3
, probe:
FAM-
TTG CCC TGA
AAG AAA GAG ATT AGG GAA
-BHQ-1
(
see
Note 4
).
′
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