plasmids and gel electrophoresis - biotechnlogy

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10 Terms

1
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What are plasmids?

  • circular extrachromosomal DNA

  • Naturally occurring

  • Replicate independently

  • Small, stable, easy to manipulate

  • Found in microbes like bacteria

<ul><li><p>circular extrachromosomal DNA </p></li><li><p>Naturally occurring </p></li><li><p>Replicate independently </p></li><li><p>Small, stable, easy to manipulate</p></li><li><p>Found in microbes like bacteria</p></li></ul><p></p>
2
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What do plasmids carry?

  • genes for metabolic activities

  • Genes are advantageous for host bacterium

  • I.e. resistance to antibiotics, heavy metals, bacteriophages

3
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Plasmids and cloning

  • plasmids are cloning vectors

  • Must have Ori: origin of replication

  • many have gene for antibiotic resistance

<ul><li><p>plasmids are cloning vectors</p></li><li><p>Must have Ori: origin of replication</p></li><li><p>many have gene for antibiotic resistance</p></li></ul><p></p>
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What process allows the study of a specific gene?

  • transformation

  • plasmid DNA is cut open: blunt or sticky ends

  • complimentary gene fragment is inserted of gene of interest

  • DNA ligase joins fragments together, forming recombinant DNA

  • plasmid is forced into cell via transformation

  • allows plasmid to independently replicate

  • allows one to gain enough material to examine

<ul><li><p>transformation</p></li><li><p>plasmid DNA is cut open: blunt or sticky ends</p></li><li><p>complimentary gene fragment is inserted of gene of interest</p></li><li><p>DNA ligase joins fragments together, forming recombinant DNA</p></li><li><p>plasmid is forced into cell via transformation</p></li><li><p>allows plasmid to independently replicate</p></li><li><p>allows one to gain enough material to examine</p></li></ul><p></p>
5
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transformation

  • introducing DNA into a bacterium from another source

  • occurs when cell takes up and expresses new genetic materia

  • new trait observed

6
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what new traits can be observed from transformation?

  • antibiotic resistance

  • frost, pest, or drought resistance

  • green fluorescence

7
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gel electrophoresis

  • used to separate DNA fragments based on size; amount of nucleotides

  • a gel with wells is soaked in a solution in a chamber between two electrodes

  • side with wells is negatively charged

  • small DNA fragments move farther and faster to positive side due to less nucleotides

<ul><li><p>used to separate DNA fragments based on size; amount of nucleotides</p></li><li><p>a gel with wells is soaked in a solution in a chamber between two electrodes</p></li><li><p>side with wells is negatively charged</p></li><li><p>small DNA fragments move farther and faster to positive side due to less nucleotides</p></li></ul><p></p>
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process of gel electrophoresis

  • wells are loaded with samples using pipette

  • gel is placed in buffer solution in a chamber with to electrodes

  • positive electron opposite to wells

  • DNA’s negative charge due to phosphate causes it to be attracted to positive side

  • smaller fragments travel faster and farther

  • larger fragments travel slower 

<ul><li><p>wells are loaded with samples using pipette</p></li><li><p>gel is placed in buffer solution in a chamber with to electrodes</p></li><li><p>positive electron opposite to wells</p></li><li><p>DNA’s negative charge due to phosphate causes it to be attracted to positive side</p></li><li><p>smaller fragments travel faster and farther</p></li><li><p>larger fragments travel slower&nbsp;</p></li></ul><p></p>
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how is gel electrophoresis measured?

  • size of fragments measured using a molecular marker → like a guide+

  • tells us size of unknown fragments 

  • allows comparison of fragment sizes from diff sources 

<ul><li><p>size of fragments measured using a molecular marker → like a guide+</p></li><li><p>tells us size of unknown fragments&nbsp;</p></li><li><p>allows comparison of fragment sizes from diff sources&nbsp;</p></li></ul><p></p>
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applications of gel electrophoresis

  • forensic: thumbprints leave unique dna sequence, can identify based on dna

  • paternity tests: matching child and possible fathers

  • medical: diagnosing genetic diseases or infections