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3MM Whatman paper |
Whatman, Dassel, Germany |
Autoclave |
Integra Biosciences, Baar, Switzland |
Benchtop gyroory incubator |
New Brunswick Scientific, Edison |
shaker for bacterium |
N.J. USA |
Bacteria incubator |
Heraeus, Hanau, Germany |
Bandelin Sonopuls HD 2070 |
South Yorkshire, England |
Cell culture flasks/dishes |
Greiner, Heidelberg, Germany |
Cell culture incubator |
Heraeus, Hanau, Germany |
CELL locate glass cover slips with grids, |
Eppendorf, Hamburg, Germany |
– Grid size 55 μm |
Cell scraper |
Corning, Wiesbaden, Germany |
Cell strainer (40μm) |
Becton Dickinson, Heidelberg, Germany |
FACS (Fluorescence-activated cell sorting) |
FACScan, Beckton Dickinson, Heidelberg |
Centrifuge |
Cytocentrifuge |
Thermo Shandon, Pittsburgh, USA |
Rotina 48 RS Table top centrifuge |
Hettich, Tuttlingen, Germany |
-Sorvall RC- 5B refrigerated superspeed |
centifuge and Biofuge 13R |
Heraeus, Hanau, Germany |
Table top centrifuge 5415C |
Eppendorf, Hamburg, Germany |
Megafuge 1.0 with Rotor BS4402/A |
Heraeus, Hanau, Germany |
Chemiluminescence detection |
-Film BioMax |
Eastman-Kodak, Rochester, USA |
Corning 125 pH meter |
Fisher Scientific, Shwerte, Germany |
Cryotubes |
Nalgene, Rochester, NY , USA |
Cytofunnel |
Shandon, Pittsburgh, USA |
Electroporation: |
Gene Pulser System with electroporation |
Bio-Rad Laboratories, Munich, Germany |
Cuvette 0.4 cm |
Flow Chamber Kit |
-Circular parallel plate |
GlycoTech, Maryland, U.S.A |
Gel electrophoresis system |
-DNA-sub cell and Mini-Sub cell System |
Gibco, Betheseida, USA |
Power PAC 2000 |
BioRad, Munich, Germany |
Irradiation of mice |
-Betatron 500A |
Siemens, Munich, Germany |
Lumat LB 9507 |
Berthold, Bad Wildbad, Germany |
Magnetic cell separation |
-MiniMACS separator |
Miltenyi, Bergisch Gladbach, Germany |
-MidiMACS separator |
Miltenyi, Bergisch Gladbach, Germany |
-MACS separation columns: LD and LS |
Miltenyi, Bergisch Gladbach, Germany |
Microscope |
-SZ40 Zoom Stereo Microscope |
Olympus, Munich, Germany |
Axioplan II-imaging Fluorescence microscope |
Zeiss, Goettingen, Germany |
Leitz Labovert inverted Microscope |
Leitz, Wetzlar, Germany |
Zeiss ID03 inverted microscope |
Zeiss, Goettingen, Germany |
Nitrocellulose membrane (0.45 μm) |
Bio-Rad Laboratories, Munich, Germany |
PCR |
-Techne TC-312 |
Kisker , Steinfurt, Germany |
Protein transfer |
-Trans-Blot SD Semi-dry Transfer cell |
BioRad, Munich, Germany |
Spectrohotometer |
-Gene Quant II |
Pharmacia Biotech, Freiburg, |
-Ultraspec® 2000 |
Germany |
Steril bank |
Heraeus, Hanau, Germany |
Steril filter |
-0.22μM |
Millipore, Eschborn, Germany |
-0.45μM |
Millipore, Eschborn, Germany |
Vortex |
Eppendorf, Hamburg, Germany |
Water bath |
Julabo Labortechnik, Seelbach, Germany |
Acetic acid glacia |
Sigma, Steinheim, Germany |
Acetone |
Roth, Karlsruhe, Germany |
Ammonium persulfate |
Fluky, Deisenhofen, Germany |
Ampicillin |
Sigma, Steinheim, Germany |
Anti-Sca-1 Microbeads |
Miltenyi, Bergisch Gladbach, Germany |
Biorad protein dye |
Biorad, Munich, Germany |
Borax |
Sigma, Steinheim, Germany |
BrdU |
Sigma, Steinheim, Germany |
β-Mercaptoethanol |
Fluka , Deisenhofen, Germany |
Bromophenol blue |
Sigma, Steinheim, Germany |
Calcium chloride |
Sigma, Steinheim, Germany |
Chlorofor |
Fluka , Deisenhofen, Germany |
Cumaric acid |
Sigma, Steinheim, Germany |
ddH2O |
Sigma, Steinheim, Germany |
DEPC |
Sigma, Steinheim, Germany |
DMSO |
Sigma, Steinheim, Germany |
EDTA, disodium, dihydrate |
Sigma, Steinheim, Germany |
Ethanol |
Merck, Darmstadt, Germany |
Ethidiumbromid |
Sigma, Steinheim, Germany |
Formaldehyd |
Merck, Darmstadt, Germany |
Forene (Isofluran) |
Abbott GmbH, Wiesbaden, Germany |
Glucose |
Sigma, Steinheim, Germany |
Glutathione sepharose |
TM |
Amersham/Pharmacia Biotech, Freiburg, |
Germany |
Glycerol |
Sigma, Steinheim, Germany |
Glycin |
Merck, Darmstadt, Germany |
Isopropylthio-β-D-Galactoside (IPTG) |
Sigma, Steinheim, Germany |
HCL |
Merck, Darmstadt, Germany |
Isobutanol |
Sigma, Steinheim, Germany |
Isopropanol |
Fluka , Deisenhofen, Germany |
Kanamycin |
Sigma, Steinheim, Germany |
Lineage cell depletion kit |
Miltenyi, Bergisch Gladbach, Germany |
Luminol |
Fluka , Deisenhofen, Germany |
Sigma, Steinheim, Germany |
MgSO4 |
Fluka , Deisenhofen, Germany |
NaN3 |
Sigma, Steinheim, Germany |
NaCl |
Merck, Darmstadt, Germany |
NaH2PO4 |
Sigma, Steinheim, Germany |
NaOH |
Roth GmbH, Karlsruhe, Germany |
Natriumacetat |
Fluka , Deisenhofen, Germany |
Paraformaldehyd |
Merck, Darmstadt, Germany |
p-Coumaric acid |
Sigma, Steinheim, Germany |
PD-10 columns |
Amersham/Pharmacia Biotec, Freiburg, |
Germany |
Phenol |
Fluka , Deisenhofen, Germany |
PKH26 Red fluorescence kit |
Sigma, Steinheim, Germany |
Polybrene |
Sigma, Steinheim, Germany |
Ponceau S |
Sigma, Steinheim, Germany |
Propedium iodide |
Sigma, Steinheim, Germany |
Protease-inhibitors |
Roche Basel, Switzerland |
Pre-stained SDS-PAGE standards |
-Broad range |
Bio-Rad Laboratories, Munich, Germany |
Retronectin |
® |
Takara Bio Inc., Otsu, Japan |
Sca-1 Isolation Kit |
Miltenyi, Bergisch Gladbach, Germany |
SDS |
Sigma, Steinheim, Germany |
STREPtactin sepharose |
TM |
IBA, Goettingen, Germany |
Protein elution buffer |
IBA, Goettingen, Germany |
TEMED |
Sigma, Steinheim, Germany |
Tritriplex III, 1,1% |
University teaching hospital Frankfurt |
Trizma HCl |
Sigma, Steinheim, Germany |
Triton X-100 |
Sigma, Steinheim, Germany |
Tween20 |
Sigma, Steinheim, Germany |
Vivapore (For concentrating protein) |
VivaScience, Stonehouse, UK |
Xylencyanol |
Sigma, Steinheim, Germany |
7-aminoactinomycin D (7-AAD) |
Sigma, Steinheim, Germany |
BSA, V solution |
Sigma, Steinheim, Germany |
Chloroquine |
Sigma, Steinheim, Germany |
DMEM, -High glucose (4.5g/l) |
Invitrogen, Karlsruhe, Germany |
DMSO |
Sigma, Steinheim, Germany |
FBS |
Gibco BRL, Paisley, Schottland |
Ficoll separating solution |
Biochrom (# L-6115), Berlin, Germany |
Gelatin |
Sigma, Steinheim, Germany |
GNF-2 |
Sigma, Steinheim, Germany |
HBSS |
Gibco, Karlsruhe, Germany |
HEPES solution |
Gibco, Karlsruhe, Germany |
Horse serum |
Gibco, Karlsruhe, Germany |
Imatinib |
Novartis, Basel Switzerland |
IMD |
Gibco, Karlsruhe, Germany |
L-Glutamine |
Gibco, Karlsruhe, Germany |
Methoculttm GF M3434 |
StemCell Technologies, Vancouver, |
Canada |
Mycosa |
Gibco, Karlsruhe, Germany |
PBS |
Gibco, Karlsruhe, Germany |
Penicillin-Streptomycin solution |
Gibco, Karlsruhe, Germany |
Retinoic acid |
Sigma, Steinheim, Germany |
Retronectin |
Takara, Shiga, Japan |
RPMI 1640 |
Gibco, Karlsruhe, Germany |
Trypan blue stain (0.4%) |
Gibco, Karlsruhe, Germany |
Trypsin EDTA, 0.25% solution |
Gibco, Karlsruhe, Germany |
rmG-CSF |
Peprotech, Offenbach, Germany |
rmSCF |
Peprotech, Offenbach, Germany |
rmIL-6 |
Peprotech, Offenbach, Germany |
rmIL-3 |
Peprotech, Offenbach, Germany |
rmGM-CSF |
Peprotech, Offenbach, Germany |
Calf intestinal phosphatase (CIP) |
NEB, Frankfurt, Germany |
Gateway LR clonase enzyme mix |
Invitrogen, Karslruhe, Germany |
Klenow-Fragment DNA-polymerase I |
NEB, Frankfurt, Germany |
Restriction endonucleases |
NEB, Frankfurt, Germany |
RNAse |
Sigma, Steinheim, Germany |
Superscript II |
Invitrogen, Karlsruhe, Germany |
T4 DNA-ligase |
NEB, Frankfurt, Germany |
Taq-DNA-polymerase |
Biosystems, Weiterstadt, Germany |
Proteinase K |
Stratagene, La Jolla, USA |
Taq-DNA-polymerase |
Biosystems, Weiterstadt, Germany |
DNTPs |
Fermentas, St. Leon-Rot, Germany |
PCR Buffer |
Fermentas, St. Leon-Rot, Germany |
MgCl2 |
Fermentas, St. Leon-Rot, Germany |
PfU turbo polymerase |
Stratagene, La Jolla, USA |
Primers |
Sigma, Steinheim, Germany |
Mouse anti-β-tubulin (Ab-4) |
Neo Markers, Asbach, Germany |
Mouse lineage panel |
Pharmingen, San Diego, CA, USA |
Rabbit anti-c-ABL (11/24) |
Santa Cruz, Heidelberg, Germany |
Rabbit anti-p-ABL-Tyr-245 |
Upstate-Biotechnology, Lake Placid, NY, |
USA |
Rabbit anti-p-ABL-Tyr-412 |
Cell Signalling, Boston, USA |
Rabbit anti-p-BCR-Tyr-177 |
Cell Signalling, Boston, USA |
Rabbit anti-BCR(C-20) |
Santa Cruz, Heidelberg, Germany |
Mouse anti- GFP |
Upstate-Biotechnology, Lake Placid, NY, |
USA |
Mouse anti-phospho-STAT5 |
Cell Signalling, Boston, USA |
Rabbit anti -STAT5 |
Cell Signalling, Boston, USA |
Rabbit anti-phospho-CrkL (Tyr207) |
Cell Signalling, Boston, USA |
Mouse anti-CrkL |
Cell Signalling, Boston, USA |
Mouse anti-phosphotyrosine (clone 4G10) |
Upstate-Biotechnology, Lake Placid, NY, |
USA |
Anti-mouse IgG-HRP |
Santa Cruz, Heidelberg, Germany |
Anti-rabbit IgG-HRP |
Santa Cruz, Heidelberg, Germany |
Mouse-anti-human-PE-LNGFR |
BD, San Jose, CA, USA |
Rat-anti- mouse-PE-Gr-1 |
BD, San Jose, CA, USA |
Rat-anti- mouse-PE-Mac-1 |
BD, San Jose, CA, USA |
Rat-anti- mouse-PE-B220 |
BD, San Jose, CA, USA |
Mouse-anti-PE-IgG 2a, K |
BD, San Jose, CA, USA |
Mouse-anti-PE-IgG 2b, K |
BD, San Jose, CA, USA |
Rat –anti-Mouse FITC-Sca-1 |
BD, San Jose, CA, USA |
Rat-anti- mouse-PE-Sca-1 |
BD, San Jose, CA, USA |
MACS buffer |
20% (w/v) BSA |
12.5 ml |
EDTA |
1mmol |
Penicillin/Streptomycin |
5ml |
Add PBS up to |
500ml |
2M CaCl2 solution |
The component was dissolved in ddH2O and sterile filtered with 0.22 μm filter and stored at – |
20°C in aliquots until use. |
2X HBS solution |
Na2HPO4 |
0.315g (1.5 mM final) |
HEPES |
19.5g (0.05M final) |
NaC |
24g (0.28M final) |
Water added up to 1200ml, three clean bottles were filled with exactly 400 ml of the solution, |
the pH to 6,95/ 7,0 / 7,05 was adjusted respectively, each bottle was filled to 500 ml, pH was |
controlled again. The buffer was filter-sterilized and stored at -20°C in aliquots. |
1x TAE Electrophoresis buffer |
Tris-Acetate |
0,04M |
EDTA |
1 mM |
Buffer W for lysis of bacteria |
Tris-HCl pH 8.0 |
100 mM |
NaCl |
150 mM |
EDTA |
1 mM |
Tween |
0.1% |
Protease inhibitor |
1x |
1X SDS gel-loading buffer |
Tris-HCl, pH 6.8 |
50mM |
SDS |
2 % (w/v) |
Glycerol |
10% (v/v) |
β-mercaptoethanol |
100mM |
Bromophenol blue |
0.1 %( v/v) |
1X SDS gel-loading buffer lacking dithiothretitol was stored at room temperature. β- |
mercaptoethanol from a 14 M stock was added just before the buffer is used. |
For protein electrophoresis |
(Invitrogen life technologies, Germany) |
NuPAGE Novex Bis-Tris |
Tris-Acetate Pre-Cast Gels |
NuPAGE Novex MES and Tris-Acetate SDS Running Buffers |
NuPAGE Transfer Buffer |
Precision plus protein (PPP) |
NuPAGE LSD Sample Buffer |
NuPAGE Reducing Agent |
NuPAGE Antioxidant |
10X TBS |
Trizma HCl, pH 7.5 |
volume was adjusted to 1 L with wate. |
TBST |
1X TBS+ 0.05% Tween 20 |
Blocking Buffer for immunoblot |
5% Carnation non-fat dry milk in TBST |
Protein transfer buffer |
TrisHCl |
48 mM |
Glycine |
Coomassie staining solution |
Coomassie brilliant blue R250 |
0.1 % (w/v) |
Methanol |
50 % (v/v) |
Glacial acetic acid |
10 % (v/v) |
Coomassie destaining Solution |
Methanol |
50 % (v/v) |
Glacial acetic acid |
10 % (v/v) |
Ponceau S staining solution |
Ponceau S |
2 % (w/v) |
Trichloroacetic acid |
30 % (v/v) |
Sulfosalicylic acid. |
30 % (v/v) |
ECLsolution I: |
1 M TrisHCl, pH 8,5 |
10ml |
90mM p-Coumaric acid (in DMSO) 0.44ml |
250mM Luminol (in DMSO) 1ml |
Water was dded up to 100ml; aliquots were made and stored in the dark at -20°C. |
ECLsolution II: |
30 % H2O2 |
64μl |
1 M TrisHCl, pH 8,5 |
10 ml |
Water was added up to 100ml, aliquat solution and store in the dark at -20°C. |
Stripping buffer |
Tris-HCl, pH8.0 |
62.5 mM |
2-mercaproethanol |
100 mM |
SDS |
2 % (w/v) |
FACS Wash Buffer |
PBS + 1%FCS + 1% NaN3 |
FACS Fixative Solution |
PBS + 2% formaldehyde solution |
Phosphate-Buffered Saline (PBS) |
NaCl |
137 mM |
KCl |
2.7 mM |
Na2HPO4 |
10 mM |
KH2PO4 |
2 mM |
The above components were dissoolved in water and adjusted pH to 7.4 with HCl. |
Tris-HCl (1M) |
121.1g of Tris base was dissolved in 800ml of water. The pH was adjusted to the desired |
10X Tris EDTA (TE) |
pH 7.4 |
100 mM Tris-Cl (pH 7.4) |
10 mM EDTA (pH 8.0) |
pH 7.6 |
100 mM Tris-Cl (pH 7.6) |
10 mM EDTA (pH 8.0) |
pH 8.0 |
100 mM Tris-Cl (pH 8.0) |
10 mM EDTA (pH 8.0) |
SDS (20% w/v) |
200 g of electrophoresis-grade SDS was dissolved in 900 ml of water. The mixture was |
heated to 68°C and stirred with a magnetic stirrer to assist dissolution. The pH was adjusted to |
7.2 by adding a few drops of concentrated HCl. The volume was adjusted to 1 liter with |
water. |
EDTA (0.5 M, pH 8.0) |
186.1 g of disodium EDTA was added to 800 ml of H2O. The mixture was stired vigorously |
on a magnetic stirrer. The pH was adjusted to 8.0 with NaOH. The disodium salt of EDTA did |
not go into solution until the pH of the solution was adjusted to ~ 8.0 by the addition of |
NaOH. |
Page 49 |
Materials |
49 |
Solutions for plasmid minipreparation |
Resuspension solution (Sol I) |
Glucose |
50 mM |
Tris-HCl pH 8.0 |
25 mM |
EDTA pH 8.0 |
10 mM |
Autoclaved and stored the solution at 4°C |
Alkaline lysis solution (Sol II) |
NaOH |
0.2 M |
SDS |
1 % (w/v) |
Sol II was prepared fresh and used at room temperature |
Neutralization solution (Sol III) |
5 M Potassium acetate |
60 ml |
Glacial acetic acid |
11.5 ml |
ddH2O 28.5 ml |
The solution was stored at 4°C and transferred to ice before use |
0.1% Diethylpyrocarbonate (DEPC) |
1 g DEPC was dissolved in 1 L water and mixed it vigiously. It let stand with loose lid |
overnight at room temperature or at 37°C for 1h and autoclaved for 15 minutes at 15 psi on |
liquid cycle. DEPC cannot be used to treat Tris buffer. |
2.3.7 Plasmids and vectors |
pCDNA 3.1 vector |
Invitrogen GmbH, Karlsruhe, Germany |
pEntr1A |
Invitrogen GmbH, Karlsruhe, Germany |
PINCO |
Retroviral hybrid vector with LTR derived from Moloney |
murine Leukemia virus. The main characteristics of this vector |
is the presence of the EBV origin of replication and the EBNA- |
1 gene and the presence of the cDNAs that encodes for the |
EGFP, controlled by a cytomegalovirus promoter.This vector |
allows high-efficiency gene transfer (Grignani et al., 1998). |
PAULO |
This vector is a modified pinco vector in which GFP is replaced |
by LNGFR |
PIDE |
This vector is based on pinco vector in which GFP is deleted and |
Hind III site can be used for cloning gene instead of GFP |
Pinco∆CMV |
Retroviral pinco based vector without any reporter gene |
Page 50 |
Materials |
50 |
pPRIBA2 |
IBA, Gottingen , Germany |
pGEX4T3 |
Amersham Biosciences, Freiburg, Germany |
2.3.8 Bacterial E.Coli Strain and genotype |
E. coli – HB101, BL21, JM83, DB 3.1, |
Invitrogen, Karlsruhe, Germany |
2.3.9 Medium for bacterium |
LB medium |
Bacto-Tryptone |
1 % (w/v) |
Bacto-Yeast-Extrac |
0.5 % (w/v) |
NaCl |
1.5 % (w/v) |
Adjuted pH to 7.4 with NaOH and autoclaved |
LB agar plates |
Bacto-Agar |
1.5 % (w/v) |
Bacterium freezing solution |
Glycerin |
65 % (v/v) |
MgSO4 |
0.1 M |
TrisHCl, pH 8.0 |
0.025M |
SOC |
Invitrogen, Karlsruhe, Germany |
2.3.10 Cell lines |
2.3.10.1 Ph+ cells |
CML blast cell lines |
In CML cells, the translocation product encodes for p210 |
BCR/ABL |
. |
BV173: Human B cell precursor leukaemia cell line, established from the peripheral blood of |
a patient with chronic myeloid leukaemia (CML) in lymphoid blast crisis. Contains the t(9;22) |
b2-a2 fusion gene. Obtained from DSMZ, Germany |
K562: Established from the pleural effusion of a patient with chronic myeloid leukaemia |
(CML) in myeloid blast crisis. Cells carry the Ph chromosome with the Bcr-Abl b3-a2 fusion |
gene. Obtained from DSMZ, Germany |
Ph+ ALL |
In Ph+ cells, the translocation product encodes for p185 |
BCR/ABL |
. |
Page 51 |
Materials |
51 |
Sup-B15: Human B cell precursor leukemia cell line, established from the bone marrow of a |
patient with acute lymphoblastic leukemia, carrying the ALL-variant (m-bcr) of the |
BCR/ABL fusion gene (e1-a2). Obtained from DSMZ, Germany |
Tom-1: human B cell precursor leukemia. Established from the bone marrow of a patient with |
refractory Ph chromosome acute lymphoblastic leukemia (ALL); described to carry the ALL- |
variant (m-BCR) of the BCR/ABL fusion gene (e1-a2) obtained from DSMZ, Germany. |
2.3.10.2 Other Cell lines |
Nalm-6: Human lymphoblastic B cell line; Ph- |
Ba/F3: IL-3 dependent murine pro B cell line established from peripheral blood; apparently |
derived from BALB/c mouse. |
32D: IL-3 dependent murine myeloid cell line established from peripheral blood. |
Rat-1: Rat fibroblast cells line |
293T: Transformed human embryonal kidney cell line |
Phoenix: Based on the 293T cell line (transformed human embryonal kidney cell line). |
Expresses the retroviral structural genes gag, pol und env. It is also known as a packaging cell |
line. The ecotropic packaging cell line delivers genes only to dividing murine or rat cells. |
All cells were obtained from the “Deutsche Sammlung von Mikroorganismen und |
Zellkulturen GmbH” (DSMZ) (German Collection of Microorganisms and Cell Cultures), |
with the exception of the ecotrophic Phoenix cells which were obtained from Nolan lab, |
Standford, USA. |
2.3.11 Medium for Cell culture |
L-glutamine |
1 % (v/v) |
FBS |
10 % (v/v) |
Penicillin/Streptomycin |
1 % (v/v) |
Added to DMEM and RPMI medium for adherent and suspension cells respectively |
Medium for 32D/Baf3 cells |
L-glutamine |
1 % (v/v) |
FBS |
10 % (v/v) |
Penicillin/Streptomycin |
1 % (v/v) |
mIL-3 |
10 ng /ml |
Added the above to RPMI medium |
Medium for Nalm-6, BV-173 and K562 |
L-glutamine |
1 % (v/v) |
Page 52 |
Materials |
52 |
FBS |
10 % (v/v) |
Penicillin/Streptomycin |
1 % (v/v |
Added the above to RPMI medium |
Medium for Tom-1 |
L-glutamine |
1 % (v/v) |
FBS |
20 % (v/v) |
Penicillin/Streptomycin |
1 % (v/v |
Added the above to RPMI medium |
Medium for Sup-B15 |
L-glutamine |
1 % (v/v) |
FBS |
20 % (v/v) |
Penicillin/Streptomycin |
1 % (v/v |
Added the above to Mycosa medium |
Medium for Sca1+/lin- mouse BM cells |
L-glutamine |
1 % (v/v) |
FBS(Hyclon) |
10 % (v/v) |
Penicillin/Streptomycin |
1 % (v/v) |
mIL-3 |
20 ng /ml |
mIL-6 |
20 ng /ml |
mSCF |
100 ng /ml |
Added the above to ISCOVE medium |
Cell freezing medium |
Solution I |
RPMI/DMEM |
70 % (v/v) |
FBS |
30 % (v/v) |
Solution II |
RPMI/DMEM |
80 % (v/v) |
DMSO |
20 % (v/v) |
2.3.12 Materials for animal experiments |
2.3.12.1 Mice |
The C57BL/6N mice (age between 6-8 weeks, all female) were purchased from Charles River |
Laboratories GmbH in Munich, Germany and served as the recipient mice for all the animal |
experiments. |
Page 53 |
Materials |
53 |
2.4 Miscellaneous |
Nucleospin |
R |
Mini plasmid DNA isolation Kit |
Macherey-Nagel, Dueren Germany |
Nucleospin |
R |
Maxi plasmid isolation kit |
Macherey-Nagel, Dueren Germany |
Gel cleaning kit |
Macherey-Nagel, Dueren Germany |
PCR cleaning kit |
Macherey-Nagel, Dueren Germany |
Qiagen gel extraction Kit |
Qiagen, Duesseldorf, Germany |
Qiagen Plasmid kit Mini, Midi and Maxi |
Qiagen, Duesseldorf, Germany |
Qiagen PCR purification Kit |
Qiagen, Duesseldorf, Germany |
Quick change site mutagenesis kit |
Stratagene, La Jolla, USA |
Endo Toxin removal kit |
Profos AG, Regensburg, Germany |
Cell proliferation (XTT) kit |
Amersham/Pharmacia Biotech, Freiburg, |
Germany |
Protein concentrator (Vivapore) |
VivaScience, Hannover, Germany |
Page 54 |
Methods |
54 |
3 Methods |
3.1 Preparation of plasmid DNA |
3.1.1 Transformation of E.coli |
A frozen vial of competent E.coli bacteria was thawed on ice. The transforming DNA (up to |
25ng per 50ul competent bacterium or 10ul ligation product) was pipetted directly to |
competent E.coli bacteria and mixed by swirling the tubes gently several time. |