3'-end modification

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This protocol is for beads, collected from ONE plate / TWO platesTWO plates / ONE plate

Содержание


NB! post-PCR operation


[править] Equipment and consumables

  • check, that you have:
» magnetic stand;
» LoBind tubes;
» rotator at 37°C;
» (highly desirable) water bath at 37°C


  • prepare Covaris S2 in post-PCR area:
» change water;
» start degassing;
» prepare 1.5ml tube holder


  • melt, vortex, spin-down and put in ice-box:
» Terminal Transferase buffer, 10x[1]: 4440µl/plate;
» CoCl2, 10x: 44µl/plate;
» Bead Linker, 50mM: 0.5µl/plate


  • shortly vortex/spin-down Terminal Transferase, 20u/µl and put it in -20°C Stratagene cooler


  • check, that you have 800µl1.6ml of 1xTEX buffer


  • prepare buffers:

TTB: 1x Terminal Transferase buffer

1 plate
Terminal transferase buffer, 10x44µl
CoCl2, 10x44µl
H2O312µl
total:400µl

2 plates
Terminal transferase buffer, 10x80µl
CoCl2, 10x80µl
H2O567µl
total:727µl



BL: 1mM Bead Linker
store at -20°C

2 libr4 libr8 libr
Bead Linker, 50mM1µl1.8µl3.6µl
LTE49µl88.2µl176.4µl
total:50µl90µl180µl





[править] Protocol

  1. declumping:
    • Covaris S2: "Covalent Declump 1";
    • pulse-spin;

  2. TTB-washing:
    • magnetic rack 1 min and remove supernatant;
    • two times:
    ○○   add 100200µl 1x TTB;
    ○○   vortex 20 sec and pulse-spin;
    ○○   magnetic rack (1 min) and remove supernatant;
    • re-suspend the beads in 178303µl 1xTTB: vortex 20 sec and pulse-spin;

  3. 3'-end modification:
    • add 2034µl #BL, vortex 10 sec and pulse-spin;
    • add 23.4µl Terminal Transferase (20u/µl) and vortex 10 sec;
    • (highly desirable) preheat tubes for 5-10 min in 37°C water bath and dry them with paper towel;
    • rotate at 37°C for 2 hours (if necessary — overnight):
    » start-time:   _________
    » finish-time:   _________

  4. two times washing:
    ○○   pulse-spin, magnetic rack (1 min) and remove supernatant;
    ○○   re-suspend the beads in 400800µl of 1xTEX: vortex 10 sec;

  5. quantitate the beads[2]




[править] Notes

  1. check, that solution is clear and w/o colour
  2. should be 4-8x105 beads/µl, or ~1.5-3.3x108 beads from each ePCR plate


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