Haynes Lab:Notebook/Jan/2013/09/27

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Plasmid Extraction and Validation of fshPCD

  • Name: fshPCD / BBa_J176002
    • Date of culture inoculation: 9/26/13
    • Results for Cultures 1 and 2: Mixture is cloudy so growth in both. Success.
    • Results for Streaks 1 and 2: Growth in both areas along zig-zag line. Bottom left of agar plate dried out. Success.
  • Sample 1: fshPCD/V0120 colony 1; BBa_J176002; part = 186 bp; vector = 3200 bp
  • Sample 2: fshPCD/V0120 colony 2; same
  • Sample 3: fshPCD/V0120 (control DNA); BBa_J176002; part = 186 bp; vector = 3200 bp

DNA Concentration Data

Plasmid OD260 OD260/280 ng/μL
1. Plasmid 1 0.186 1.859 185.784
2. Plasmid 2 0.138 1.827 138.008


Restriction Digest Table

  • Checked plasmid minipreps with EcoRI/PstI digests
Reagent Volume Expected:
1. Plasmid 1 = 186 bp, 3200
2. Plasmid 2 = 186, 3200
Today's gel
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 3.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 8.5
  15 μL --> 37°C/ 15 min.

Conclusion: All samples had a band just under the 5000 bp band (the brightest top band), which was probably the 3200 bp vector. Also, the expected part base pair was 186 bp, and the band in the picture is slightly above the 200 bp band, but all three samples including the control DNA had the band at the same location, so overall, the restriction digest was successful. However, additional help about the two bands around the 1500 bp location that appeared in samples 1 and 2 but not in the control DNA will be received before continuing. After talking with the professor, a possible explanation is that this band was supercoiled undigested plasmid DNA. It was decided to submit samples 1 and the control DNA for DNA sequencing.

DNA Sequencing Samples

  • Submitted to DNASU on: '10/8/13'
  • Order Number: 8215
  1. fshPCD - forward primer 'P0001' 186/186 matches. 100% alignment across the sequence.
  2. fshPCD - reverse primer 'P0002' 186/186 matches. 100% alignment across the sequence.
  3. fshPCD-control DNA - forward primer 'P0001' 186/186 matches. 100% alignment across the sequence.
  4. fshPCD-control DNA - reverse primer 'P0002' 186/186 matches. 100% alignment across the sequence.




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