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
table 7: 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 |
|
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