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Practical molecular biology 8.10-.12.2012. PD Dr. Alexei Gratchev Prof Dr. Julia Kzhyshkowska Prof. Dr. Wolfgang Kaminski. Assistants. Tina Fuchs Martin Hahn Amanda Mickley Illya Ovsiy. Course structure. 8.10 Plasmids, restriction enzymes, analytics 9.10 Genomic DNA, RNA

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practical molecular biology 8 10 12 2012

Practical molecular biology 8.10-.12.2012

PD Dr. Alexei Gratchev

Prof Dr. Julia Kzhyshkowska

Prof. Dr. Wolfgang Kaminski

assistants
Assistants
  • Tina Fuchs
  • Martin Hahn
  • Amanda Mickley
  • Illya Ovsiy
course structure
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)
literature
Literature
  • Current protocols in molecular biology
  • Molecular Cloning: A Laboratory Manual, Third Edition by Sambrook
  • www.methods.info
plasmids restriction enzymes analytics
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.

plasmids are essential instruments of molecular biology
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
essential vector elements
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)

optional plasmids elements
Optional plasmids elements
  • Multiple cloning site
  • Promoter for cloned sequence
  • Reporter gene
  • Tag
  • Regulatory sequences
important plasmid information
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.
restriction enzymes endonucleases
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
restriction enzymes endonucleases15
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.
slide16

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.

slide17

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.

restriction enzymes endonucleases18
Restriction enzymes (endonucleases)
  • Creating genomic and cDNA libraries
  • Cloning DNA molecules
  • Studying nucleotide sequence
  • Generating mutated proteins
plasmids restriction enzymes analytics19
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)

plasmid preparation stage 1
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

preparation of the lysate
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.

possible methods for isolation
Possible methods for isolation

Ethanol or Isopropanol precipitation

Silica matrix bind-wash-elute procedure

Density gradient centrifugation

precipitation quick and dirty
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
silica matrix columns
Silica matrix columns

Apply lysate on the column

Wash the column

Elute the plasmid

Precipitate

Advantages

Disadvantages

  • High purity of the plasmid
  • Fast
  • Expensive
gradient centrifugation
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
concentration measurement
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.

gel electrophoresis of plasmid dna
Gel electrophoresis of plasmid DNA

Selection of agarose concentration

Plasmid on an agarose gel