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Emulsion PCR

Protocol Notes  Comments

NB! pre-PCR operation

Prepare

• Covaris S2:
  • change water;
  • start degassing;
  • prepare post-PCR tube holder;
• clean vortex and picofuge in post-PCR room;
• reserve PCR machine (1x 96-well plate per standard reaction) & prepare 4mm aluminum plate for cover;
• check programs on XStream:
  •   "emulsion" program:
  • setting: Pip
  • speed up: 5 (mid-range)
  • speed down: 1 (lowest)
  • volume: 5600µl
  •   "dispensing" program:
  • setting: Dis
  • speed up: 1 (lowest)
  • speed down: 1 (lowest)
  • volume: 150µl
• check, that there are enough 10ml Combitips, 5ml wide Combitips & 200µl wide tips;
• decide, how to dilute sequencing library to 50pg/µl & fill dilution protocol (preferable >2µl aliquots);
• (i) melt, (ii) vortex, (iii) spin-down and (iv) put in freeze-box:
  • 10x PCR buffer (560µl/plate);
  • 25mM dNTP mix (800µl/plate);
  • 100µM ePCR Primer 1 (2.5µl/plate);
  • 500µM ePCR Primer 2 (35µl/plate);
  • library stock;
  • BBS (Bead Block Solution, 200µl/plate);
• shortly vortex/spin-down & put in ice-box an aliquot of AmpliTaq Gold DNA polymerase 5u/µl (600µl per plate);
• equipment:
  • 0.01g balance;
  • ULTRA-TURRAX Tube Drive;
  • picofuge;
  • vortex;
  • magnetic stand;
  • plate sealer or adhesive covers;
• check, that you have:
  • powder-free gloves;
  • LoBind tubes & tips;
  • tube holders: 50ml, 2ml, 0.5ml;
  • working portions of buffers:
    
    

    1 plate 2 plates 4 plates 1M MgCl2 140µl 280µl 560µl 1xTEX bufer 160µl 320µl 0,7ml LTE (1X Low TE Buffer) 250µl 500µl 1ml BBS (Bead Block Solution) 160µl 320µl 640µl

• label according to the library list (one per library):
  • 96-well PCR plates: #lib;
  • ULTRA-TURRAX tubes: oil phase, do not label;
  • wide tubes for aqueous PCR phase: #lib;
  • 0,5ml LoBind tubes:
      #lib 1ng/µl - 1ng/µl dilutions of seq. libraries,
      #lib 50pg/µl - 50pg/µl dilutions of seq. libraries,



• prepare buffers:



◊   #OP (oil phase) in 50ml tube, 9ml per plate, fresh weekly, store at NT
NB! the order of reagents corresponds to their densities, so, it is possible to correct a mistake of pipetting

1 plate 2 plates 4 plates Emulsion stabilizer 2, 1.06g/ml 0.106g 100µl 0.201g 190µl 0.39g 370µl Emulsion stabilizer 1, 0.97g/ml 0.437g 450µl 0.829g 855µl 1.62g 1.665ml Emulsion oil, 0.858g/ml 8.15g 9.45ml 15.49g 17.96ml 30.15g 34.965ml Total, 0.865g/ml: 10ml 19ml 37ml

• mix, 2 min vortex;
• put 7.8g / 9ml in each of #lib TURRAX tube (for 1 plate)



◊   #PCR1/10 (10µM ePCR primer 1): vortex to mix well, store in ice;

1 plate 2 plates 4 plates ePCR primer 1, 100µM 2.5µl 5µl 10µl LTE 22.5µl 45µl 90µl Total: 25µl 50µl 100µl




◊   aqueous PCR phase

1 plate 2 plates 4 plates H2O   3.294ml 4x824µl 6.589ml 7x941µl 13.178ml 10x PCR buffer 1x 560µl 1.12ml 2.28g / 2.24ml 3x 747µl dNTP mix, 25mM 3.5mM 784µl 1.568ml 3.22g / 3.136ml 4x 784µl MgCl2, 1M 25mM 140µl 280µl 0.59g / 560µl #PCR1/10, 10µM 40nM 22.4µl 44.8µl 89.6µl ePCR Primer 2, 500µM 3µM 33.6µl 67.2µl 134.4µl Total:   4.834ml 9.668ml 19.338ml

• mix;
• aliquot 4.8ml / 4.8g in wide tubes;
• keep on ice;


◊   dilution of seq. libraries 50pg/µl LTE (0.5ml tubes #lib)
_____µl of _____ng/µl stock  + _____µl of LTE => 1ng/µl
2µl of 1ng/µl stock  +  38µl of LTE  ⇒  40µl of 50pg/µl solution

E-beads charging

1. should be done in advance when kit comes:
   • vortex P1 Beads (1 min) & pulse-spin;
   • aliquot 160µl of beads (~1.3x109) into 1.7ml tubes;
2. wash P1 beads:
   • magnetic rack (20 sec) & remove supernatant;
   • re-suspend in 160µl of BBS;
   • vortex (30 sec) & pulse-spin;
   • 2 times declumping:
       ◊  ◊   Covaris S2: "Bead Block Declump";
       ◊  ◊   pulse-spin;
   • magnetic rack (1 min) & remove supernatant;
   • re-suspend in 160µl of TEX:
   • vortex (30 sec); pulse-spin & store in ice-box;
3. prepare to ePCR:
   • check that PCR machine is free;
   • prepare XStream and Combitips;
   • et 5 min on ULTRA-TURRAX Tube Drive;



NB! §§4-6 should be done without any delay for each PCR plate
4. prepare PCR aqueous phase:
   • add to aqueous PCR phase & keep on ice (per plate):
       ◊   AmpliTaq Gold, 5u/µl: 600µl
       ◊   template #lib, 50pg/µl:
          

     concentration [pg/ml] volume [µl] 50 5.6 75 8.4 100 11.2

   • vortex beads (30 sec) and pulse-spin;
   • Covaris S2: 2x "Covalent Declump 1 - beads in 1xTEX"/2;
   • pulse-spin & add all (160µl) beads into PCR aqueous phase;
   • mix carefully (do not vortex!);
5. prepare emulsion:
   • place TURRAX tube onto ULTRA-TURRAX Tube Drive, twist to lock;
   • draw 5.6ml using Xstream: 1(!) click;
   • attach Combitip to TURRAX tube;
   • start 5 min run of ULTRA-TURRAX Tube Drive, wait for 5 sec;
   • deliver all content: first click - small excess; second - main part; and twice more - the rest;
6. ePCR:
   • smoothly deliver 150µl in wells of PCR plate (should be ~all wels);
   • seal the plate;
   • put into PCR machine & start run;
7. after ePCR:
   • put in the box pre-PCR tube holder;
   • check, that emulsion remains stable;
   • store PCR plate at +4°C for several days;

Notes

• ePCR program:
  1. 0.5°C to 95°C
  2. 95°C 5:00
  3. 0.5°C to 93°C
  4. 93°C 0:15
  5. 0.5°C to 62°C
  6. 62°C 0:30
  7. 0.5°C to 72°C
  8. 72°C 1:15
  9. go to 3, 39 times
  10. 72°C 7:00
  11. 0.5°C to 4°C
  12. 4°C hold
  
  set: "lid tracking with 2°C";
  
  
  • we did not see any differences when using "MJ research mashine" or "9700 PCR mashine from ABI"



• library
  • stock library should be stored at -20°C at concentration >5ng/µl;
  • normal dilutions of sequencing library: 50pg/µl (~2.5x10⁹ molecules/ePCR plate);



• stock of P1 beads is ~8x10⁶beads/µl;



• it is important to use a proper type of the sealing tape: the recommended in the protocol "MicroAmp™ Clear Adhesive Film (Applied Byosystems, #4306311)" is a bad choice. The best in our hands is film from TESA (Tesafilm #2826775, ~5€ per roll);



• rough estimate: ePCR fails if plate have more than 3 wells with broken emulsion;
• sealing programs: