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Line 307: |
Line 307: |
| |-bgcolor=#009be1 | | |-bgcolor=#009be1 |
| |+ align="top, left"|other reagents | | |+ align="top, left"|other reagents |
- | | Name||Suplier|| Cat. No. ||Reference | + | | Name||Suplier|| Cat. No. |
| |- | | |- |
- | |align="right"| Matrix Reagent Reservoirs || Thermo Scientific || 8094 || || | + | |align="right"| Matrix Reagent Reservoirs || Thermo Scientific || 8094 |
- | |- | + | |- |
- | |align="right"| cell culture flasks 75cm2 || TPP || 90076 || || | + | |align="right"| cell culture flasks 75cm2 || TPP || 90076 |
| |- | | |- |
- | |align="right"| Dual-Luciferase® Reporter Assay System || Promega || E1910 || || | + | |align="right"| Dual-Luciferase® Reporter Assay System || Promega || E1910 |
| |- | | |- |
- | |align="right"| white 96-well plates, lumitrac200 || Greiner || 655075 || || | + | |align="right"| white 96-well plates, lumitrac200 || Greiner || 655075 |
| |- | | |- |
- | |align="right"| Rapid DNA Ligation Kit 40 || Roche || 11635379001 || || | + | |align="right"| Rapid DNA Ligation Kit 40 || Roche || 11635379001 |
| |- | | |- |
- | |align="right"| Quick ligation kit || NEB || M2200 S || || | + | |align="right"| Quick ligation kit || NEB || M2200 S |
| |- | | |- |
- | |align="right"| 15 cm petri dishes || BD falcons|| || || | + | |align="right"| 15 cm petri dishes || BD falcons|| |
| |- | | |- |
- | |align="right"| Master Mix Phusion, HF || Finnzymes || || || | + | |align="right"| Master Mix Phusion, HF || Finnzymes || |
| |- | | |- |
- | |align="right"| 6x Loading Dye || Fermentas || || || | + | |align="right"| 6x Loading Dye || Fermentas || |
| |- | | |- |
- | |align="right"| Penicilin Streptomycin || Gibco || || || | + | |align="right"| Penicilin Streptomycin || Gibco || |
| |- | | |- |
- | |align="right"| T4 DNA ligase || Fermentas || || || | + | |align="right"| T4 DNA ligase || Fermentas || |
| |- | | |- |
- | |align="right"| FuGENE 6 || Roche || || || | + | |align="right"| FuGENE 6 || Roche || |
| |- | | |- |
- | |align="right"| Proteinase K || Qiagen || || || | + | |align="right"| Proteinase K || Qiagen || |
| |- | | |- |
- | |align="right"| poly-L-lysine || Sigma || || || | + | |align="right"| poly-L-lysine || Sigma || |
| |- | | |- |
- | |align="right"| Benzonase (5kU)|| Clontech || || || | + | |align="right"| Benzonase (5kU)|| Clontech || |
| |- | | |- |
| |} | | |} |
Materials
Kits
table 1: Used kits.
Kits | Supplier | Catalog Number
|
HiSpeed® Plasmid Maxi Kit (25) | QIAGEN | 12663
|
CompactPrep Plasmid Maxi Kit (25) | QIAGEN | 12763
|
RNeasy® Mini Kit (25) | QIAGEN | 74106
|
QIAquick® Miniprep Kit (250) | QIAGEN | 27106
|
QIAquick® Gel Extraction Kit (250) | QIAGEN | 28706
|
QIAquick® Nucleotid Removal Kit (250) | QIAGEN | 28306
|
QIAquick® PCR Purification Kit (250) | QIAGEN | 28106
|
GeneMorph® II EZC Clone Domain Mutagenesis Kit | Stratagene | 200552-5
|
Marker
table 2: Used DNA ladders for gel electrophoresis.
Name | Supplier | Range
|
GeneRuler™ High Range DNA Ladder | Fermentas | 10,171-48,502 bp
|
GeneRuler™ 1 kb Plus DNA Ladder | Fermentas | 75-20,000 bp
|
Enzymes
table 3: Used NEB restriction enzymes.
Name | Number/ID | NEB buffer1 | NEB buffer2 | NEB buffer3 | NEB buffer4 | BSA
|
AflII | 27 | 50% | 100% | 25% | 100% | x
|
AgeI | 1 | 100% | 50% | 10% | 75%
|
ApaI | - | 25% | 50% | 0% | 100% | x
|
AseI | not | 75% | 100% | not |
|
AvrII | - | 100% | 100% | 50% | 100% |
|
BamHI | 2 | 75% | 100% | 100% | 100% | x
|
BamHI | - | 75% | 100% | 100% | 100% | x
|
BclI | - | 50% | 100% | 100% | 75% |
|
BglI | - | 50% | 75% | 100% | 50% |
|
BglII | 13 | 10% | 75% | 100% | 10% |
|
BsrGI | - | 25% | 100% | 10% | 100% | x
|
DpnI | 6 | 100% | 100% | 75% | 100% |
|
DpnI | - | 100% | 100% | 75% | 100% |
|
EcoRI | - | 100% | 100% | 100% | 100% |
|
EcoRV | - | 50% | 75% | 100% | 50% | x
|
FseI | - | 100% | 75% | 0% | 100% |
|
HindIII | - | 10 50% | 100% | 10% | 50% |
|
HindIII | - | 50% | 100% | 10% | 50% |
|
KpnI | 11 | 100% | 75% | 0% | 50% | x
|
MfeI-HF™ | - | 75% | 50% | 10% | 100% |
|
NarI | 26 | 100% | 75% | 75% | 100% |
|
NcoI | 12 | 100% | 100% | 100% | 100% |
|
NdeI | - | 75% | 100% | 75% | 100% |
|
NheI | - | 100% | 100% | 10% | 100% | x
|
NotI | 14 | 0% | 50% | 100% | 25% |
|
NsiI | - | 10% | 75% | 100% | 25% |
|
PciI | - | 50% | 75% | 100% | 50% | x
|
PstI | 15 | 75% | 75% | 100% | 50% |
|
SacI | 16 | 100% | 50% | 10% | 100% |
|
ScaI | 18 | not | not | 100% | not |
|
SfcI | 19 | 75% | 50% | 10% | 100% | x
|
SfcI | - | 75% | 50% | 10% | 100% | x
|
SpeI | - | 75% | 100% | 25% | 100% | x
|
SphI | - | 100% | 100% | 50% | 100% |
|
SspI | - | 50% | 100% | 50% | 50% |
|
XbaI | 23 | 0% | 100% | 75% | 100% | x
|
XbaI | 56 | 0% | 100% | 75% | 100% | x
|
XhoI | 24 | 75% | 100% | 100% | 100% | x
|
XmaI | 25 | 25% | 50% | 0% | 100% | x
|
NEBuffers are color-coded (NEBuffer 1-yellow, NEBuffer 2-blue,
NEBuffer 3-red, NEBuffer 4-green) and supplied as 10X
Bacteria
table 4: Used E. coli strains.
Name | Purpose | Reference
|
DH5α | Amplification of Plasmids | Invitrogen
|
SCS110 | Amplification of unmethylated Plasmids | Stratagene
|
Top10 | Amplification of Plasmids | Invitrogen
|
Media and Antibiotics
- LB (10 g tryptone, 5 g yeast extract, 10 g sodium chloride, ad 1 L ddH2O)
table 5: Used antibiotics.
Antibiotic | Final Concentration | Solvent
|
Ampicillin | 100 µg/ml | ddH2O
|
Chloramphenicol | 25 µg/ml | 100% Ethanol
|
Kanamycin | 50 µg/ml | ddH2O
|
Primers & Oligos
Please find our Primers & Oligos on an extra subpage.
Instruments
table 6: Used instruments.
Description | Name | Firm
|
Table Centrifuge | Microfuge® 18 Centrifuge | Beckman CoulterTM
|
Table Centrifuge | MicrofugeR 22® Centrifuge | Beckman CoulterTM
|
Centrifuge | Allegra X-12 | Beckman CoulterTM
|
Shaker | VORTEXGENIER 2 | Scientific Industries, SITM
|
Heating plate (magnet stirrer) | MR Hei-Standard | Heidolph
|
Heatblock | QBD4 | Grant
|
Heatblock (shakeing function) | Thermomixer comfort | Eppendorf
|
PCR-machine | MyCyclerTM thermo cycler | BioRad
|
UV-chamber | Transluminator | Vilber Lourmat
|
Scale (fine) | PioneerTM PA114C | OHAUS
|
Scale | PioneerTM PA4101C | OHAUS
|
Fridge | KTP 1750 Premium | Liebherr
|
Freezer | GP 1366 Premium | Liebherr
|
-86 Freezer | -86°C Ultralow Freezer | NUAIRETM
|
Hood | Tischabzug | Wesemann® Laboreinrichtung
|
Draw-off pump | Vacuhand control | Vacubrand
|
Incubator | HT Multitron Version 2 | INFORS
|
Incubator (cell culture) | HERAcell 150 | Thermo electron coorperation
|
Hood (cell culture) | HERAsafe | Thermo electron coorperation
|
Plate reader | Infinite M200 | TECAN
|
FACS | Cytomics FC 500 MPL | Beckman CoulterTM
|
Microscope | CKX41 | Olympus
|
Computer | | Sun Microsystems
|
UV/Visivle Spectrometer | Ultrospec 3300 pro | Amersham Biosciences
|
Photometer | NanoDrop® ND-1000 Spectrophotometer | peQLab Biotechnologie GmbH
|
Photometer | NanoVueTM | General Electric
|
Ice Machine | MF22 | SCOTSMAN®
|
Gel electrophoresis chamber | Mupid®-One | Advance
|
Pipetting robot | QIAcubeTM | QIAGEN
|
Wide field microscope | Nikon Eclipse 90i | Nikon
|
PCR machine | MyCycler thermal cycler | BIO-RAD
|
Real-time PCR machine | StepOnePlusTM Real-time PCR System | Applied BiosystemsTM
|
Cell lines
Used Cell Lines.
Cell line | Tissue of origin | Transfection
|
HeLa P4 | epithelial cervical cancer, human | easy |
|
HEK 293T | embryonic kidney cells, human | easy |
|
Flp-In™ T-REx™ 293 | embryonic kidney cells, human | easy |
|
Hep G2 | hepatocellular carcinoma, human | medium |
|
Hepa 1-6 | liver hepatoma, mouse | medium |
|
HuH7 | hepatocarcinoma, mouse | medium |
|
primary hepatocytes | liver, mouse | hard |
|
Plasmids
Used Plasmids.
Name | Use | Sequence Information | Reference
|
pcDNA5 | expression of shRNAs | CMV_shRNA_polyA | [http://tools.invitrogen.com/content/sfs/manuals/pcdna5frtto_man.pdf Invitrogen] |
|
pcDNA6/TR | amplification of Tet Repressor | CMV_TetR_polyA | [http://tools.invitrogen.com/content/sfs/vectors/pcdna6tr_map.pdf Invitrogen] |
|
PsiCheck2 | cloning of tuning construct | HSV_hRluc/SV40_hFluc | [http://www.promega.com/catalog/catalogproducts.aspx?categoryname=productleaf_1602 Promega] |
|
pBSU6 | virus packaging (ssDNA) | ITR (AAV2) | Addgene |
|
pTRUF3 | virus packaging (dsDNA) | ITR (AAV2) | |
|
pBSH1 | virus packaging (ssDNA) | ITR (AAV2) | |
|
pSB1A3 | 3A assembly cloning | Ampicillin Resistance | [http://partsregistry.org/Part:pSB1A3 Parts Registry] |
|
pSB1K3 | 3A assembly cloning | Kanamycin Resistance | [http://partsregistry.org/Part:pSB1K3 Parts Registry] |
|
pSB1C3 | 3A assembly cloning | Chloramphenicol Resistance | [http://partsregistry.org/Part:pSB1C3 Parts Registry] |
|
Other
other reagents
Name | Suplier | Cat. No.
|
Matrix Reagent Reservoirs | Thermo Scientific | 8094
|
cell culture flasks 75cm2 | TPP | 90076
|
Dual-Luciferase® Reporter Assay System | Promega | E1910
|
white 96-well plates, lumitrac200 | Greiner | 655075
|
Rapid DNA Ligation Kit 40 | Roche | 11635379001
|
Quick ligation kit | NEB | M2200 S
|
15 cm petri dishes | BD falcons |
|
Master Mix Phusion, HF | Finnzymes |
|
6x Loading Dye | Fermentas |
|
Penicilin Streptomycin | Gibco |
|
T4 DNA ligase | Fermentas |
|
FuGENE 6 | Roche |
|
Proteinase K | Qiagen |
|
poly-L-lysine | Sigma |
|
Benzonase (5kU) | Clontech |
|
|