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.)