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Research facilities

Cloning & Purification Facility

Location
Science Campus
Einsteinweg 55
2333 CC Leiden

Cloning & Purification Facility

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

Culturing

  • Infors HT  Ecotron and Minitron dry shakers (temperature range 15 – 40 °C)
  • Gyrotory waterbath shakers (New Brunswick)

DNA manipulations

  • T100 Thermal cycler 2x (BioRad)
  • Eppendorf concentrator 5301
  • Radioactive 32P labeling (D-Lab and B-lab)

Quantification protein/DNA

  • Qubit Quantitation (Life technology)
  • Eppendorf Biophotometer (BioRad assay, BCA assay, A260, A280)
  • ChemiDoc XRS and GS-800 densitometer (BioRad): UV (EtBr gels)

-80 C storage for bacterial strains and purified proteins

  • VIP U101 tabletop freezer (New Brunswick)
  • Thermo ultra low floor model freezer

When you are interested to have a project in the CPF you can contact:

Dr. Nora Goosen
n.goosen@chem.leidenuniv.nl
phone: 071 527 4773
office 03.29

 

Geri Moolenaar
moolenaa@chem.leidenuniv.nl
phone: 071 527 4772
office 03.31