The Effects of Pollutants on puc-19 Plasmid DNA

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The Effects of Pollutants on puc-19 Plasmid DNA

• Lena Sutten
• Devin Altman

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Hypothesis

• Environmental pollutants, diesel, oil, and
  exhaust pipe scrapings, cause damage to puc-19
  plasmid DNA.

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DNA

• Puc-19 is a Bacterial DNA.
• Supercoiled shape vs. Double Helix structure

Double Helix DNA vs. Plasmid DNA
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Plasmid DNA puc-19

• Easy to work with
• Small
• Supercoiled
• Easy to detect damage (nicked)
• Contains charged particle () ion

www.geocities.com
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Gel Electrophoresis

http//www.life.uiuc.edu/molbio/geldigest/uvbox.ht
ml

• http//www.life.uiuc.edu/molbio/geldigest/casting.
  html

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Gel ElectrophoresisWhat is going on here?

• F q E
• Apply Voltage
• Phosphate Ion (DNA) travels through gel
• qE vf

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Results1st gel run of about 20 minutes of
exposure time.

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Results2nd Gel run of about an hour exposure
time.

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Gel ElectrophoresisNormal Looking Gel
The smaller fragments of DNA will travel faster
than the larger pieces of DNA. Thus, damaged DNA
(smaller fragments) will be further down the gel.
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Conclusion

• No conclusive evidence
• Gel pictures do not show any damage
• Need to use glycerol dye to hold pollutant in
  wells
• Use lower voltage, 50 V vs. 100 V
• Practice makes perfect!

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What Will We Do Next?

• Gain Access to Weyerhaeuser Co.s lab in Federal
  Way
• Cultivate Plasmid DNA to see if pollutants can
  penetrate the cell membrane.
• Increase pollutant exposure time.
• Decrease pollutant concentration.
• Test air particulates and SF5CF3.

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The Millipore protocol for isolation of plasmid
DNA from bacteria using the Montage Plasmid
Miniprep96 Kit with the MultiPROBEâII Liquid
Handling System is shown below. Website
http//www.millipore.com/analytical/pubdbase.nsf/
docs/TN10060000.html
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