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3'-End modification

Equipment and consumables Protocol Notes  Comments

NB! post-PCR operation. This protocol is for beads, collected from ONE plate / TWO platesTWO plates / ONE plate

Equipment and consumables

• vortex;
• magnetic stand;
• powder-free gloves;
• LoBind tubes & tips;
• rotator at 37°C;
• (optional) 37°C; water bath;


• (optional) Covaris S2:
  • change water;
  • start degassing;
  • prepare post-PCR tube holder;


• (i) melt, (ii) vortex, (iii) spin-down and (iv) put in ice-box:
  • 10x Terminal Transferase buffer (NB! check, that solution is clear & w/o colour) 44µl/plate;
  • 10x CoCl₂ 44µl/plate;
  • 50mM Bead Linker 0,5µl/plate;
• shortly vortex/spin-down and put in -20°C cold-box Terminal Transferase (20u/µl);



• prepare #TTB (1x Terminal Transferase buffer):

  1 libr 2 libr 4 libr 10x Terminal transferase buffer 44µl 88µl 176µl 10x CoCl2 44µl 88µl 176µl H2O 312µl 624µl 1.248ml Total: 400µl 800µl 1.6ml

  1 libr 2 libr 4 libr 10x Terminal transferase buffer 82.5µl 165µl 330µl 10x CoCl2 82.5µl 165µl 330µl H2O 585µl 1.17ml 2.34ml Total: 750µl 1.5ml 3ml

• prepare #BL (1mM Bead Linker):

  2 libr 4 libr 8 libr 50mM Bead Linker 1µl 1.8µl 3.6µl LTE 49µl 88.2µl 176.4µl Total: 50µl 90µl 180µl

  2 libr 4 libr 8 libr 50mM Bead Linker 1.4µl 2.8µl 5.6µl LTE 68.6µl 137.2µl 274.4µl Total: 70µl 140µl 280µl

Protocol

1. declumping:
   • Covaris S2: "Covalent Declump 1";
   • pulse-spin;
2. TTB-washing:
   • magnetic rack 1 min & remove supernatant;
   • two times:
     ◊  ◊   add 100200µl 1x TTB;
     ◊  ◊   vortex 20 sec & pulse-spin;
     ◊  ◊   magnetic rack (1 min) & remove supernatant;
   • re-suspend the beads in 178303µl 1x TTB: vortex 20 sec & pulse-spin;
3. 3'-end modification:
   • add 2034µl #BL, vortex 10 sec & pulse-spin;
   • add 23.4µl Terminal Transferase (20u/µl) & vortex 10 sec;
   • (optional) preheat tubes in 37°C water bath & dry them with paper towel;
   • rotate at 37°C for 2 hours:
     start-time:   _________
     finish-time:   _________
4. two times washing:
   ◊  ◊   pulse-spin, magnetic rack (1 min) & remove supernatant;
   ◊  ◊   re-suspend the beads in 400800µl of TEX: vortex 10 sec;
5. quantitate the beads

Notes

§ 3

• if necessary, rotate at 37°C overnight;

§ 5

• should be ~5x10⁵ beads/µl, ~2x10⁸ total from each ePCR plate;