Restriction enzyme digest

Digest DNA with restriction enzyme DpnII, which cleaves at GATC cut sites. All DNA from the three sample pools will be digested in 50 µL reactions.

Reaction setup:

reagent volume per reaction (µL) Mastermix for 3 reactions (µL)
DNA 25
Buffer 5 15.5
H2O 19 58.9
DpnII 1 3.1
Total: 50 77.5

Add 25 µL of Mastermix to each tube of pooled DNA (each containing 25 µL DNA, see prev. entry)

Digest:

Set thermocycler for:

  • 3 hours at 37°C
  • 20 min. at 65°C
  • Hold at 15°C

(Frozen overnight - bead clean and gel to run tomorrow)


June 1, 2016

Post-digest bead clean:

  1. Add 90 µL Ampure XP beads to 50 µL digested DNA (1.8:1 vol. beads:DNA)
  2. Incubate at room temperature for 5 min.
  3. Place on magnetic stand for 5 min.
  4. Remove supernatant into waste container, leaving ~5 µL behing
  5. Add 200 µL freshly made 80% ethanol (on magnetic stand)
  6. Incubate 30 sec. then remove supernatant to waste container
  7. Add 200 µL feshly made 80% ethanol (on magnetic stand)
  8. Incubate 30 sec. then remove supernatant to waste container
  9. Remove any residual ethanol using pipet
  10. Let dry for 5 min., not longer than 10 min.
  11. Resuspend beads in 30 µL H2O, pipet up and down
  12. Incubate at room temperature for 5 min.
  13. Place on magnetic stand for 5 min.
  14. Transfer 28 µL supernatant to destination tube

Gel of digested, bead-cleaned DNA:

1% agarose gel run @ 100V for 40 min. 3 µL each sample + 1 µL loading dye; 3 µL HyperLadder 50bp.

  • BC = Bleached clade C pooled samples
  • NC = Not-bleached clade C pooled samples
  • ND = Not-bleached clade D pooled samples

Gel 1

Result: smear of DNA is visible in all three pooled samples between ~200-1000bp. Digest appears to have been successful.