Biomod/2011/TUM/TNT/LabbookA/Immobilizing structures with biotin-oligos for TIRF

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Contents

Prerequisites

  • PBS Buffer 1x, filtered with 0.2 μm filter
  • NeutrAvidin (diluted to 0.05 mg/ml with filtered PBS)
  • BSA (diluted to 5 mg/ml with filtered PBS)
  • Biotin-Oligos 0028_MS (100 nM, Biotin-TEG-TAGAGCCCAGACACGTTAACCAGAG)
  • Your Sample with the oligo antisense sequence (diluted 1:1000 - 1:10000 with 0.2 μm filtered sample-buffer)

Procedure

  1. flush with NeutrAvidin (35-40µl)
  2. incubate 10 min
  3. flush with PBS two times (100µl each)
  4. wait for 5 min
  5. flush with PBS
  6. flush with BSA (75µl)
  7. incubate 15min
  8. flush with PBS two times
  9. wait for 5 min
  10. flush with PBS
  11. flush with Biotin-oligos (60µl)
  12. incubate 30 min
  13. flush with probe-buffer two times
  14. wait for 10 min
  15. flush with sample-buffer (without DNA-binders)
  16. flush with sample (without DNA-binders)
  17. incubate for 20 min
  18. flush with sample-buffer two times (with DNA binders)

Old Procedure and Prerequisites

Until 29.09.11, a slightly different procedure was used for the immobilization of structures for TIRF:

  • buffers were not filtered
  • BSA was used in a concentration of 0.5 mg/ml instead of 5 mg/ml
  • NeutrAvidin was incubated for 5-10 min instead of 10 min (step 2.)
  • BSA was incubated for 10-15 min instead of 15 min (step 7.)
  • samples were incubated for 30 min instead of 20 min (step 17.)