Research facilities

Cloning & Purification Facility

Location: Gorlaeus Building
Einsteinweg 55
2333 CC Leiden

The Cloning and Protein purification Facility (CPF) provides expertise and assistance in molecular biology techniques. We train and help researchers, BSc and MSc students of the Leiden Institute of Chemistry with their research projects including:

Constructions of plasmid clones, mutants, fusion constructs etc.
Construction of specific E.coli strains
Protein production in E.coli
In vitro (cell free) protein production
Centrifugation instructions
Protein purifications
Development of enzymatic assays
Analysis of protein-DNA interactions

Cloning

We can help with the design of expression plasmids to overproduce your gene of interest in E.coli. We have a large collection of vector plasmids for the generation of fusion constructs with different types of tags (GST/His/MBP/Chitin/Trx/DsbA) to facilitate the purification and/or solubility of the protein. Genes can be isolated via PCR or can be designed for De Novo gene synthesis (in collaboration with BaseClear).
Cloning procedures involve classical Restriction Enzyme digestion/ ligation and Gibson assembly. Desired mutations can be introduced in the gene of interest using site-directed mutagenesis.
The facility has a large collection of Restriction Enzymes, polymerases, ligase, kinase etc. permanently in stock as well as kits for plasmid isolations and DNA purifications.

Protein Expression and Purification

The facility has a large collection of E.coli strains for protein expression. Upon request additional mutations can be introduced in the production strain. E.coli cells can be cultured on small and large scale at different temperatures. Upon request proteins can also be produced in a cell-free system.
For the protein purifications the CPF houses 2 Äkta Starts and 1 Äkta purifier. If desired the tag can subsequently be removed from the protein of interest using proteases. Different methods to improve the solubility of the protein are available. Protein concentrations can be determined using the Bradford assay, the BCA assay or using the Qubit.
We can also store the purified proteins in a safe way for future use.

Locations

The CPF is located in the renovated part of the LMUY building in the Gorlaeus Lab. The department has two labs, one is dedicated for cloning and DNA manipulations (room 03.30), the other (room 03.34) for protein purification and biochemistry.

The facility also houses a D-lab (03.24) for the analysis of 32P-labeled samples and a dark room (03.38) to facilitate work with light sensitive labels.
When needed the Facility provides a working place in one of the offices where you have wireless access via the university provided networks. In our shared room you can meet the other CPF participants and exchange ideas.

Centrifugation

Beckman Coulter Ultracentrifuge Optima L90K with fixed angle rotors 70.1 Ti and 70 Ti and swing out SW41 rotor
Sorvall RC6plus with Fiberlite F10-6x500y (500 ml) F13-14x50cy (50 and 15 ml), SS-34 (50 and 12 ml) rotors
Tabletop centrifuge Eppendorf 5810 with swing out A-62 rotor, in cold room, 3220xg
Tabletop centrifuge Eppendorf 5702 with swing out rotor A4-38, 3000xg
Tabletop centrifuge Heraeus 1S-R with swing out rotor TTH400, with cooling, 4618xg
Eppendorf Minispin with PCR-strip rotor F-55-16-5-PCR
Eppendorf 5424 with FA-45-24-11 rotor (1.5 and 2.0 ml), 20.000xg
Eppendorf 5430R, with FA-45-24-11 HS rotor (1.5 and 2.0 ml), with cooling, 30.000xg

Protein purification

GE Healthcare Äkta Start 2x
Amersham Pharmacia Äkta Explorer
Pharmacia Gradifrac 2x
Circulation cooler Prolabo F250 2x
Sonics Vibracell VCX 750: tapered microtip (3 mm) and replaceable tip (13 mm)

Electrophoresis

BioRad PowerPac Basic and 3000 power supplies
BioRad Mini-Protean Tetra Cell: acrylamide mini gels (vertical)
Hoefer Mighty Small: acrylamide mini gels (vertical)
Horizontal electrophoresis units small, midi and large (Agarose gels)
Vertical electrophoresis adjustable single sequencer 20-42 cm SU-400-20 (CBS Scientific): long (denaturing) acrylamide gels