Today Sam walked me through the process of using Agilent 2100 Bioanalyzer kit to assess DNA integrity (bp length) via fluorescence signal. Unique aspect of using this kit/analyzer is that it only requires 1ul of sample, at DNA concentrations between 0.5-50 ng/ul.

Steps:

  • Sam removed kit reagents from fridge to sit at room temperature for 30 minutes
  • Prepared new gel-dye mix as per kit instructions. Prepared mix can be held at 4C for up to 4 weeks. All dye vials should be protected from light.
  • Combined my sample reps into -A vial, e.g. transferred DNA from 1-B and 1-C to vial 1-A.
  • Walked to Seeb lab in the 1st floor of the Marine Science building. Materials I needed: DNA samples, new DNA chip, prepared gel-dye mix, DNA marker & ladder from kit, DI water (for chip wells without my samples), 10ul pipette, 10ul pipette tips.
  • Followed kit instructions – loaded gel, marker, ladder, samples, and DI water onto chip. Samples 1-8 were loaded into the wells of the same number. Wels 9-12 were loaded with DI water.
  • Turned computer on, started DNA 1200 series software. NOTE: computer has login password – make sure I have that for future uses.
  • Inserted chip into analyzer, closed lid carefully.
  • For Assay Selection file, selected dsDNA -> DNA 1200 series 11.xsy
  • For Destination file location, navigated to Roberts Lab folder
  • Selected Start

The software displays data in real-time with fluoresence on y-axis, and time on the x-axis. Smaller DNA fragments “elute” faster, and likewise longer fragments take longer. Each sample well also has small (50bp) and large (17,000) standards/markers. We were hoping my DNA fragments would be long/as intact as possible. Here are some screen-shots of each fluoresence/time plot, along with the software’s calculated bp/concentrations:

Screenshot of all plots; my samples are labeled Sample 1 through Sample 8. Samples 9-12 are just DI water. Samples 1-7 look somewhat consistent, but Sample 8 looks very weird. Note different y-axis range.

image

Screenshots of the bp/concentration break-down:

Sample #1 image

Sample #2 image

Sample #3 image

Sample #4 image

Sample #5 image

Sample #6 image

Sample #7 image

Sample #8 image

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Assessing larval DNA integrity
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