Practical molecular biology 8.10-.12.2012

1. Practical molecular biology 8.10-.12.2012 PD Dr. Alexei Gratchev Prof Dr. Julia Kzhyshkowska Prof. Dr. Wolfgang Kaminski

2. Assistants • Tina Fuchs • Martin Hahn • Amanda Mickley • Illya Ovsiy

3. Course structure • 8.10 Plasmids, restriction enzymes, analytics • 9.10 Genomic DNA, RNA • 10.10 PCR, real-time (quantitative) PCR • 11.10 Protein analysis IHC • 12.10 Flow cytometry (FACS)

4. Groups

5. Literature • Current protocols in molecular biology • Molecular Cloning: A Laboratory Manual, Third Edition by Sambrook • www.methods.info

6. Plasmids, restriction enzymes, analytics Plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. Vector – a carrier (plasmid or other type) used for bringing target DNA fragment into a host cell.

7. Vector types

8. Plasmids are essential instruments of molecular biology • Cloning and sequencing of DNA and cDNA fragments • Generation of genomic and cDNA libraries • Expression of recombinant proteins • Generation of mutant proteins • Analysis of regulatory sequences • Gene targeting

9. Essential vector elements • Origin of replication • Antibiotic resistance gene (Amp, Kan, Tet, Chl) • (Multiple cloning site) Map of pOTB7 vector showing Chloramphenicol resistance gene (CMR), replication origin (ORI) and multiple cloning site (MCS)

10. Optional plasmids elements • Multiple cloning site • Promoter for cloned sequence • Reporter gene • Tag • Regulatory sequences

11. Important plasmid information • Replication origin defines the host bacteria: ColE1 replication origin is required for E.coli • Replication origin may define the number of plasmid copies per bacterial cell • Bacteria may lose recombinant plasmid during cultivation due to the absence of partitioning system (par). Naturally occurring plasmids contain par that ensures that every bacterial cell contains the plasmid.

12. Selection of the plasmid vectorCopy number

13. Selection of the plasmid vectorPurpose of use

14. Restriction enzymes (endonucleases) • Cut specific DNA sequence • Protect bacteria from phage infection by digesting phage DNA after injection • Cellular DNA is protected by methylation that blocks restriction enzyme activity • Restriction enzyme (RE) means restricts virus replication • Endonucleases are enzymes that produce internal cut called as cleavage in DNA molecule

15. Restriction enzymes (endonucleases) • Presence of RE was postulated in 1960 by W.Arber • The first true RE was isolated in 1970 by Smith, Nathans and Arber. In 1978 they were awarded the Nobel Prize for Phylsiology and Medicine. • RE remain indispensible from molecular cloning and sequencing.

16. Restriction enzymes (endonucleases) Type I enzymes cut at a site that differs, and is located at least at at least 1000 bp away, from their recognition site. Type II enzymes recognize sites of 4-8 nucleotides and cleave DNA at the same site Type III enzymes recognize two separate non-palindromic sequences that are inversely oriented. They cut DNA about 20-30 base pairs after the recognition site.

17. Restriction enzymes (endonucleases) Type I enzymes cut at a site that differs, and is located at least at at least 1000 bp away, from their recognition site. Type II enzymes recognize sites of 4-8 nucleotides and cleave DNA at the same site Type III enzymes recognize two separate non-palindromic sequences that are inversely oriented. They cut DNA about 20-30 base pairs after the recognition site.

18. Restriction enzymes (endonucleases) • Creating genomic and cDNA libraries • Cloning DNA molecules • Studying nucleotide sequence • Generating mutated proteins

19. Plasmids, restriction enzymes, analytics Gel electrophoresis is a technique used for the separation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein molecules using an electric current applied to a gel matrix. Ethidium bromide stained agarose gel of total RNA (1-3) and DNA ladder (M)

20. Plasmid preparation stage 1 Plasmid-containing bacteria are cultivated in liquid media, supplemented with the antibiotics for 18 h at 37°C with intensive shaking Cells are harvested by centrifugation

21. Preparation of the lysate 3 solutions strategy Resuspend in hypotonic buffer with RNase (buffer P1) Lyse bacteria using NaOH/SDS solution (buffer P2) Neutralize NaOH and precipitate proteins using NaAc buffer (buffer P3) Plasmid can be isolated from obtained lysate using various strategies.

22. Possible methods for isolation Ethanol or Isopropanol precipitation Silica matrix bind-wash-elute procedure Density gradient centrifugation

23. Precipitation “quick and dirty” Also known as mini prep Ethanol is added to the lysate Obtained sample incubated for 30 min DNA is collected by centrifugation Advantages Disadvantages • Cheap • Fast • Small amounts of DNA • Poor purity, not sufficient for applications like transfection and in vitro translation • Concentration of the plasmid can not be determined photometrically

24. Silica matrix columns Apply lysate on the column Wash the column Elute the plasmid Precipitate Advantages Disadvantages • High purity of the plasmid • Fast • Expensive

25. Gradient centrifugation Mix lysate with CsCl solution Add EtBr Centrifuge in the ultracentrifuge for 12-36h Collect the plasmid Precipitate Advantages Disadvantages • The very best plasmid purity • Relatively cheap • Slow • Expensive equipment is needed • High concentrations of EtBr

26. Concentration measurement Photometric measurement of DNA concentration UV 260 nm Conc=50xOD260 Important! Photometric measurement of DNA concentration can not be applied for “quick and dirty” plasmids, because of the presence of RNA rests.

27. Gel electrophoresis of plasmid DNA Selection of agarose concentration Plasmid on an agarose gel

28. Questions?