Team:UCLA/Notebook/Spider Silk Genetics/23 June 2015

iGEM UCLA




6/23/2015

Streptavidin Binding Test

  • Today we are testing if the streptavidin beads are binding properly to the biotinylated DNA.
  1. Prepared 10 uL of magnetic beads with 1 uL of 5 uM initiator.
    • Incubated 35 min.
    • Washed 2x with 100 uL 0.5x BW buffer
  2. Prepare contructs A-B and C-Term (C-T) separately,
    • Prepared each in 10 uL reactions with 0.5 uL T7 ligase.
    • Incubate each at 25 C for 10 minutes.
  3. Combined A-B and C-T to form A-B-C-T
    • Incubate 25 C for 10 minutes.
  4. Mix A-B-C-T onto the prepared beads.
    • Incubate at 25 C for 10 minutes with rotation.
  5. Saved the supernatant, then added 1 uL of 5 uM initiator to it.
  6. Incubate RT for 10 minutes.
  7. After bead ligation, eluted using 0.01% Tween-20 at 95 C for 5 minutes with occasional mixing.
  8. Ran 25 uL PCR using 1 uL of eluate as template.
  9. Saved all samples in -20 C.