transfection
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TRANSFECTION
Ingrida Olendraitė
Molecular biologist
Vilnius university
2013
TRANSFECTION
[trans-fek´shәn]
artificial introduction of foreign DNA into cultured eukaryotic cells
AIM:
to introduce one of the most popular methods of cloning
OUTLINE
Competent cells Vector Thermal shock
Growing of colonies Expression of proteins
Conclusion
COMPETENT CELLS
cells are said to be competent, when they are more likely to incorporate foreign DNA
cell walls are altered so that DNA can pass through more easily
calcium chloride / electroporation and heat shock E. coli
ELECTROPORATION
VECTOR
a Latin word meaning "carrier“ Vectors are pieces of DNA that are used to transfer
genes into a host cell pUC or pET are commonly used plasmids as a
cloning vector in E.coli
THERMAL SHOCK
occurs when a thermal gradient causes different parts of a cell to expand by different amounts
the effect of subjecting a cell to a higher temperature than that of the ideal body temperature of the organism from which the cell line was derived
COLONIES
Luria Bernati (LB) media Agar Antibiotic (exempli gratia: ampicilin)
COLONIES
LB – agar media Transformed E. coli 37 oC, 6-10h.
COLONIES
Re-seed the most
productive colonies
OD600 0,4-0,6
Induction (e.g. IPTG)
EXPRESSION OF PROTEINS
Polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS) buffer.
CONCLUSION
Transfection is simple and very useful method, often used by molecular biologists
RESOURCES
“European molecular biology laboratory” http://www.embl.de/pepcore/pepcore_services/protein_expression/ecoli/
https://www.youtube.com/watch?v=Hf0sen7bJ6A http://dwb4.unl.edu/Chem/CHEM869N/
CHEM869NLinks/www.brunel.ac.uk/depts/bio/project/genome/moltec/vectors/puc19.htm
Great thanks to squad of departament of biological DNA modification at institute of biotechnology
Have a good day!
Live long and prosper (S.C.)
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