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Lernmaterialien für Introduction to Molecular Biology an der Universität für Bodenkultur Wien

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TESTE DEIN WISSEN

Watson and Crick?

Lösung anzeigen
TESTE DEIN WISSEN
  • the composition of DNA varies from one species to another
  • total amounts of pyrimidine equal to the amounts of purine: A+G=C+T
  • after Frnklin and Gosline made a xray diffraction analysis they comined the information
    • Nucleotides from a linear chein, linked by phospodiester bonds
    • DNA froms a holical double strand
    • antiparallel organization
    • minor and major groove
    • 1 double helix turn - 10 bp
Lösung ausblenden
TESTE DEIN WISSEN

Identification of recombinant clones (electroporation)?

Lösung anzeigen
TESTE DEIN WISSEN
  • after electroporation
    • many Ecoli will have taken up one plasmid
    • very few will contain 2 oder more
    • many Ecoli cells without plasmid DNA are present
  • each plasmid molecule carries the selection marker gene (pET11a -> ampicillin)
  • on a medium containing ampicillin only Ecoli that carry a plasmid are able to grow
Lösung ausblenden
TESTE DEIN WISSEN

Histone Code?

Lösung anzeigen
TESTE DEIN WISSEN

numerous possible combinations

  • various effects on gene expression
  • highly dynamic and reversible
  • interaction between chromatin remodelling, modification histone variants -> impacts on gene expression


Lösung ausblenden
TESTE DEIN WISSEN

Cloning vectors?

Lösung anzeigen
TESTE DEIN WISSEN
  • small DNA molecule within a cell
  • physically seperated from chromosomal DNA 
  • replicates indipendently
  • commonly in bacteria, sometimes in archae and eukaryotic organisms
  • carry genes that benefit the survival of the organism (Antibiotic resistence)
  • plamids and be transmitted from one bacterium to another via three mechanisms
    • transformation
    • transduction
    • conjugation
  • naked DNA -> do not encode genes
  • size of the plasmid varied from 1 to 200 kbp
Lösung ausblenden
TESTE DEIN WISSEN

DNA sequencing?

Lösung anzeigen
TESTE DEIN WISSEN
  • 2 types of DNA sequencing
    • Dideoxy cycle DNA sequencing:
      • for sequencing single clones
    • High troughput, next generation DNA sequencing:
      • for genome and transcriptome sequencing
Lösung ausblenden
TESTE DEIN WISSEN

Gel Electrophoresis?

Lösung anzeigen
TESTE DEIN WISSEN
  • Gene materials:
    • argarose gel (routine application, longer than 200 bp)
    • polyacrylamide (short DNA fragments
  • DNA wanders to anode because of phosphate groups
  • sieve effect letzs long DNA fragments migrate more slowly
Lösung ausblenden
TESTE DEIN WISSEN

Regulation of DNA Replication?

Lösung anzeigen
TESTE DEIN WISSEN
  • Initiation of replication requires cis-acting elements: Origin of Replication (ORI)
  • preferentially A-T rich sequences - facilitated DNA helix opening
  • Initiator Proteins associate with ORI
  • promotes interaction with DNA Helicase
  • DNA Primase adds RNA primer
  • replication machinery starts with DNA replication in both directions
  • replication fork ist established
Lösung ausblenden
TESTE DEIN WISSEN

Pre PCR mispriming?

Lösung anzeigen
TESTE DEIN WISSEN
  • can happen though unspecific annealing
  • very high concentration of free primers favors unspecific annealing
  • even at room temperature Taq Polymerase has some activity and the formation of primer dimers is likely which leads to a massive loss of active primers
  • self extension of primer dimer intead of extension on the template DNA
  • Solution: small organic molecule attaches to the polymerase and is destroyed by heat so that the polymerase can take its place
Lösung ausblenden
TESTE DEIN WISSEN

Hybridization?

Lösung anzeigen
TESTE DEIN WISSEN
  • tm is the temperature at which half of the DNA is double stranded and half of the DNA is single stranded
  • annealing temperature should be 4 degrees under tm
  • we want a annealing temperature as high as possible in order to prevent unspecific annealing
Lösung ausblenden
TESTE DEIN WISSEN

PCR History

Lösung anzeigen
TESTE DEIN WISSEN
  • first proposed by Khorana in 1970
  • made public by Kary Mullis in 1985
  • method of the year in 1989
  • nobel prize in 1993
Lösung ausblenden
TESTE DEIN WISSEN

PCR Components?

Lösung anzeigen
TESTE DEIN WISSEN
  • Template
  • 2 Primers per template
  • Buffer (buffer substance, salt)
  • thermostable Polymerase (thermophillic bacteria - Thermus aquaticus, pyrococcus furiosus)
Lösung ausblenden
TESTE DEIN WISSEN

Usage of primers of cDNA

Lösung anzeigen
TESTE DEIN WISSEN
  • Oligo dT
    • + primes on poly A+mRNA
    • + crude RNA preps
    • -Rere RNA can be underpresented
  • Random pdN6
    • +large cDNA library
    • - cDNA can be small in size
    • - poly A- template can also be reverse transcribed
  • Gene specific
    • + cDNa library is enriched for specific mRNA
    • cDNA libraried can be made for specific applications, cloning all antibody encoding
Lösung ausblenden
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Q:

Watson and Crick?

A:
  • the composition of DNA varies from one species to another
  • total amounts of pyrimidine equal to the amounts of purine: A+G=C+T
  • after Frnklin and Gosline made a xray diffraction analysis they comined the information
    • Nucleotides from a linear chein, linked by phospodiester bonds
    • DNA froms a holical double strand
    • antiparallel organization
    • minor and major groove
    • 1 double helix turn - 10 bp
Q:

Identification of recombinant clones (electroporation)?

A:
  • after electroporation
    • many Ecoli will have taken up one plasmid
    • very few will contain 2 oder more
    • many Ecoli cells without plasmid DNA are present
  • each plasmid molecule carries the selection marker gene (pET11a -> ampicillin)
  • on a medium containing ampicillin only Ecoli that carry a plasmid are able to grow
Q:

Histone Code?

A:

numerous possible combinations

  • various effects on gene expression
  • highly dynamic and reversible
  • interaction between chromatin remodelling, modification histone variants -> impacts on gene expression


Q:

Cloning vectors?

A:
  • small DNA molecule within a cell
  • physically seperated from chromosomal DNA 
  • replicates indipendently
  • commonly in bacteria, sometimes in archae and eukaryotic organisms
  • carry genes that benefit the survival of the organism (Antibiotic resistence)
  • plamids and be transmitted from one bacterium to another via three mechanisms
    • transformation
    • transduction
    • conjugation
  • naked DNA -> do not encode genes
  • size of the plasmid varied from 1 to 200 kbp
Q:

DNA sequencing?

A:
  • 2 types of DNA sequencing
    • Dideoxy cycle DNA sequencing:
      • for sequencing single clones
    • High troughput, next generation DNA sequencing:
      • for genome and transcriptome sequencing
Mehr Karteikarten anzeigen
Q:

Gel Electrophoresis?

A:
  • Gene materials:
    • argarose gel (routine application, longer than 200 bp)
    • polyacrylamide (short DNA fragments
  • DNA wanders to anode because of phosphate groups
  • sieve effect letzs long DNA fragments migrate more slowly
Q:

Regulation of DNA Replication?

A:
  • Initiation of replication requires cis-acting elements: Origin of Replication (ORI)
  • preferentially A-T rich sequences - facilitated DNA helix opening
  • Initiator Proteins associate with ORI
  • promotes interaction with DNA Helicase
  • DNA Primase adds RNA primer
  • replication machinery starts with DNA replication in both directions
  • replication fork ist established
Q:

Pre PCR mispriming?

A:
  • can happen though unspecific annealing
  • very high concentration of free primers favors unspecific annealing
  • even at room temperature Taq Polymerase has some activity and the formation of primer dimers is likely which leads to a massive loss of active primers
  • self extension of primer dimer intead of extension on the template DNA
  • Solution: small organic molecule attaches to the polymerase and is destroyed by heat so that the polymerase can take its place
Q:

Hybridization?

A:
  • tm is the temperature at which half of the DNA is double stranded and half of the DNA is single stranded
  • annealing temperature should be 4 degrees under tm
  • we want a annealing temperature as high as possible in order to prevent unspecific annealing
Q:

PCR History

A:
  • first proposed by Khorana in 1970
  • made public by Kary Mullis in 1985
  • method of the year in 1989
  • nobel prize in 1993
Q:

PCR Components?

A:
  • Template
  • 2 Primers per template
  • Buffer (buffer substance, salt)
  • thermostable Polymerase (thermophillic bacteria - Thermus aquaticus, pyrococcus furiosus)
Q:

Usage of primers of cDNA

A:
  • Oligo dT
    • + primes on poly A+mRNA
    • + crude RNA preps
    • -Rere RNA can be underpresented
  • Random pdN6
    • +large cDNA library
    • - cDNA can be small in size
    • - poly A- template can also be reverse transcribed
  • Gene specific
    • + cDNa library is enriched for specific mRNA
    • cDNA libraried can be made for specific applications, cloning all antibody encoding
Introduction to Molecular Biology

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