Assessing larval DNA integrity
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.
- 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. It looks like the long bp internal standard may have been erroneously high?