This document discusses the process of transfecting eukaryotic cells with foreign DNA. Transfection involves making cells competent to uptake DNA, using a vector like a plasmid to carry the DNA, and applying a thermal shock to get the DNA into the cells. The transfected cells are then grown on media plates containing antibiotics to select successfully transformed colonies that express the target protein, which can be verified through gel electrophoresis. Transfection is a simple but useful method employed by molecular biologists to introduce cloned genes into cells.

1. TRANSFECTION
Ingrida Olendraitė
Molecular biologist
Vilnius university
2013

2. TRANSFECTION
 [trans-fek´shәn]
 artificial introduction of foreign DNA into
cultured eukaryotic cells

3. AIM:
 to introduce one of the most popular methods
of cloning

4. OUTLINE
 Competent cells
 Vector
 Thermal shock
 Growing of colonies
 Expression of proteins
 Conclusion

5. 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

6. ELECTROPORATION

7. 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

8. 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

9. COLONIES
 Luria Bernati (LB) media
 Agar
 Antibiotic (exempli gratia: ampicilin)

10. COLONIES
 LB – agar media
 Transformed E. coli
 37 oC, 6-10h.

11. COLONIES
 Re-seed the most
productive colonies
 OD600 0,4-0,6
 Induction (e.g. IPTG)

12. EXPRESSION OF PROTEINS
 Polyacrylamide gel electrophoresis (PAGE) in
sodium dodecyl sulfate (SDS) buffer.

13. CONCLUSION
 Transfection is simple and very useful method,
often used by molecular biologists

14. RESOURCES
 “European molecular biology laboratory”
http://www.embl.de/pepcore/pepcore_services/prot
ein_expression/ecoli/
 https://www.youtube.com/watch?v=Hf0sen7bJ6A
 http://dwb4.unl.edu/Chem/CHEM869N/CHEM869N
Links/www.brunel.ac.uk/depts/bio/project/genome/
moltec/vectors/puc19.htm

15. Great thanks to squad of departament of biological
DNA modification at institute of biotechnology

16. Have a good day!
Live long and prosper (S.C.)