Infrastructure of the Cell Biology Laboratory (CBL)
The CBL is divided into 3 functional parts:
1) a General Area for storage, preparing media, cleaning, incubating and finally a small desk for computer work.
2) a Molecular Biology Area for performing standard techniques as PCR, electrophoresis, restriction digestion, photodocumentation, electroporation and microbiological work.
3) a Cell Culture Area exclusively for cultivating and working with human cell lines.
General Area
The general reagents and the labware are stored in a small board, for refrigeration (4 °C) and freezing (- 20 °C) we use a refrigerator/freezer combination (Bosch, KSR 390 & GSD 320).
Storage boards
Refrigerator (Bosch)
Hazardous reagents (corrosive, flammable) are stored in a plywood safety cabinet (SciMatCo, SC2036) below the extraction hood in the Molecular Biology Area (Mobelab). Cell lines are stored in liquid nitrogen in a Dewar (Chart, XC43/28) in the gas house out side of the laboratory, as well the CO2 cylinders.
Extraction hood with safety cabinet below
Gas house at the back side of the CBL: CO2 cylinder, Dewar (in the box for protection against dust). In the back a rack for storage of plastic-ware
For preparing the media we have a small working bench next to the storage board. For adjusting the pH we use a multiparametric station (Orion Thermo, 5 Star) and a hot plate magnetic stirrer (Corning, PC-420D). On the opposite side we have an antivibration table for the fine balance (AND, HM-120) and a usual balance (Setra, SI-4100S).
pH-meter (Orion) & hot plate stirrer (Corning)
Antivibratoric table with balance (Setra & AND)
Bacterial (37 °C) or yeast (30 °C) cells are incubated in a medium sized incubator (Binder, BD 115) which bears an orbital shaker (IKA, KS260 basic) inside for shaking incubations. Additionally we have an orbital shaker for shaking incubations at room temperture (Thermo, MaxQ-2000). The usual distilled water we have to buy; this is also used to produce 18 MΩ/cm ultrapure water for critical molecular biology or cell culture purpose (Millipore, MilliQ Biocel). The autoclave (Yamato, SN-500C) for sterilizing is easy to use and fulfils the basic safety regulations.
Incubator (Binder) with shaker inside (IKA)
MilliQ Biocel (Millipore) and Autoclave (Yamato)
The cleaning area is very simple with a washbasin and a wall mounted drying rack. The office area is right next to the entrance/exit and consists of a small desk for the responsible of the lab as well as for the students. Nothing special here, too.
The glassware is from Schott (bottles), the rest is from Pyrex. Pipettes are mainly from Eppendorf (Research and Easypet). The plastics are from Corning, Eppendorf (tubes, tips), Nalgene (bottles) or Heathrow Scientific (racks).
Standard reagents are from Merck, Sigma-Aldrich, J.T. Baker, Research Organics. Media, antibiotics and reagents/kits for molecular biology are also from Bio-Rad, Difco, Invitrogen, enzymes we obtain mainly from Fermentas.
Safety
Several safety aspects were considered when constructing the CBL.
At the exit is located the safety area which contains:
- an emergency stop (Siemens) which, when pressed, interrupts all electricity and the gas line (butane)
- an emergency shower and the eye shower (Bradley), which have an independent water system (450 l)
- a CO2-extinguisher of 4.5 kg
- a first aid box
- all safety signs are fluorescent so they are even after a loss of electricity still visible (DIN 4844). Additionally fluorescent markers advise before obstacles (moedel).
Other small safety details are:
The Bunsen burner has a small pilot and can not be turned off directly but only at the principal outlet (to avoid keeping gas under pressure in the tube).
The water tap can be opened without using (possibly contaminated) hands but with the arm (for not contaminating the tap).
The surfaces of the working benches have a small border (to avoid dropping down of a liquid after an accidental release).
Safety area
Safety area (by day)
Safety area (by night)
Molecular Biology
The objective of this area is constructing the molecular biology tools to study protein-protein interactions. For this we construct expression vectors for the cell culture (in-vivo studies), for microbial expression (co-immunoprecipitation) and for yeast-2-hybrid test.
Additionally, we construct bacterial expression vectors for other purposes co-operating with other working groups. Actually, we are working on a rhamnolipid expression system.
Thus we do standard cloning techniques in the molecular biology area. With the Thermocycler (Techne, TC-3000) we do the PCR, for agarose gel electrophoreses we use a medium sized chamber (Owl, B2) with a strong power supply (Major Science, MP-3AP), which can also be used for Western blotting. The photodocumentation (Major Science, Digimage DI-01) can be used with white epi-illumination or UV-transillumination (Major Science, MUV21) for ethidium bromide stained gels. For safety reasons the UV-light of the transilluminator turns off automatically when the access door of the photodocumentation system is opened. Next to this we have a small microwave (Daewoo, KOR-6NDB) for solubilizing agarose.
Thermocycler (Techne), electrophoresis chamber (Owl) and power supply (Major Science)
Photodocumentation system with UV-transilluminatior (Major Science)
On the opposite side we have some small equipment: An electroporator for transformation of bacteria, yeast and cell lines (Eppendorf, 2510), a multipurpose microcentrifuge (Eppendorf, 5415D) and a photometer (Mecasys, Biopop) and a cooling drybath (FinePCR, ALB6400).
The initial objective of the cell culture area was studying the protein trafficking (the interaction between the inflammatory mediator P-selectin and the endocytotic factor Numb3). Now we are establishing several techniques for cytotoxicity studies, especially for active compounds isolated from medicinal plants.
For culturing the cell lines we use a small CO2-incubator (MCO-5AC, Sanyo), which has a copper-enriched stainless steel interior which inhibits possible bacterial contaminants. The required CO2 is delivered by "Infra Gas" (#32050, Type Coleman). The manipulation of the cells occurs within a class II biological safety cabinet (NuAire, Nu-425-400) and for observing the cells we use a stereomicroscope (EMZ-5, Meiji).
For studying the protein-trafficking of the cells or cytotoxic effects of secondary compounds from medicinal plants, we have an epi-fluorescence microscope (Zeiss, Axioskop 40FL) and the documentation will be done with a camera (Lumenera, Infinity 3-1UC) connected to the software Image J. To minimise vibrations during photo capturing, the microscope is placed on a antivibratoric table. Additionally, we have a bigger refrigerated centrifuge (Eppendorf, 5804R) for separating or collecting cells. Due to the reduced space of the CBL, this is also used for preparing electrocompetent cells (E. coli) and doing Midi-preps.
CO2-incubator (Sanyo) and Stereomicroscope (Meiji)
Refrigerated centrifuge (Eppendorf)
Biological Safety Cabinet (NuAire) and Refridgerator (Bosch)
Epi-fluorescence microscope (Zeiss) with Infinity 3-1UC camera (Lumenera)
Special conical tubes, which resist high centrifugation forces, are needed for the Eppendorf centrifuge with the fixed angle rotor "F-34-6-38":
