EPCR amplification

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NB! pre-PCR operation


[править] Preliminary

  • estimate the scale of ePCR reaction:

one 96-well PCR plate gives ~150-330mln of washed and enriched beads, so for SOLiD 3+

Deposition chamber
[wells]
Beads to load
[mln per well]
Expected number of MP seq. reads [mln]Number of PCR plates depending on
- number of wells -
- 1 -- 2 -- 3 -- 4 -
1700450-500471013
412580-9011-1.523
85535-400.50.511
* it is difficult to prepare emulsion and process beads for less than 1/2 of the PCR plate


  • check that you know the mean length of sequencing libraries (mean length after library amplification)
» ~155bp for fragment libraries
» ~300bp for mate-pair libraries


  • check that you have:
» 0.01g balance
» ULTRA-TURRAX Tube Drive
» picofuge
» vortex
» magnetic stand
» adhesive tape for PCR plates
» powder-free gloves
» LoBind tubes and tips
» tube holders: 50ml, 2ml, 0.5ml


  • clean vortex and picofuge in the pre-PCR room


  • prepare Covaris S2 in the pre-PCR room:
» change water
» start degassing
» prepare 1.5ml tube holder


  • reserve necessary number of PCR machines
» (only for MJ Research PCR machines) prepare 4mm aluminum cover plates


  • check programs on XStream pipette:
emulsion
settingPip
speed up5 (mid-range)
speed down1 (lowest)
volume5600µl


dispensing
settingDis
speed up1 (lowest)
speed down1 (lowest)
volume150µl


  • check, that there are enough:
» 10ml Combitips: 1 /plate
» 5ml wide Combitips: 1 /plate
» 200µl wide tips: 1 /plate


  • check that you have enough buffers:

1 plate2 plates4 plates
1M MgCl2140µl280µl560µl
1x TEX bufer160µl320µl0,7ml
LTE (1x Low TE Buffer)250µl500µl1ml



  • melt, vortex, spin-down and put in ice:
library stock 
PCR buffer, 10x560 µl/plate
dNTP mix, 25mM800 µl/plate
ePCR Primer 1, 100µM2.5 µl/plate
ePCR Primer 2, 500µM35 µl/plate
BBS (Bead Block Solution)200 µl/plate


  • prepare aliquots of AmpliTaq Gold DNA polymerase 5u/µl (600µl per plate): shortly mix by finger tapping, spin-down and put back on -20°C in a Stratagene cooler


  • label with library names (one per plate):
» 96-well PCR plates: "#lib"
» ULTRA-TURRAX tubesfor oil phase: do not label
» wide tubes for aqueous PCR phase: "#lib"
» 0.5ml LoBind tubes for dilutions of seq. libraries: "#lib, 0.5nM"


[править] Buffers preparation

[править] 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 pipetting mistake:

ρ [g/ml]1 plate2 plates4 plates
Emulsion stabilizer 21.060.106g
100µl
0.201g
190µl
0.39g
370µl
Emulsion stabilizer 10.970.437g
450µl
0.829g
855µl
1.62g
1.665ml
Emulsion oil0.8588.15g
9.45ml
15.49g
17.96ml
30.15g
34.965ml
total0.86510ml19ml37ml

  • mix, 2 min vortex (NB! the swirl should come from the very bottom of the tube)
  • aliquot 7.8g / 9ml in TURRAX tubes
  • keep on room temperature


[править] #PCR1/10

10µM ePCR primer 1: vortex to mix well, store in ice

1 plate2 plates4 plates
LTE22.5µl45µl90µl
ePCR primer 1, 100µM2.5µl5µl10µl
total25µl50µl100µl


[править] aqueous PCR phase

 1 plate2 plates4 plates
H2O 3.294ml
4x824µl
6.589ml
7x941µl
13.178ml
PCR buffer, 10x1x560µl1.12ml2.28g / 2.24ml
3x 747µl
dNTP mix, 25mM3.5mM784µl1.568ml3.22g / 3.136ml
4x 784µl
MgCl2, 1M25mM140µl280µl0.59g / 560µl
#PCR1/10, 10µM40nM22.4µl44.8µl89.6µl
ePCR Primer 2, 500µM3µM33.6µl67.2µl134.4µl
total 4.834ml9.668ml19.338ml

  • mix
  • aliquot 4.8ml / 4.8g in wide "#lib" tubes
  • keep on ice




[править] ePCR

  • should be done in advance upon arrival of the kit:
» vortex P1 Beads (1-3 min) and pulse-spin
» aliquot 160µl of beads (~1.3x109) into 1.7ml tubes
» store at 4°C


  1. decide, how to dilute sequencing library in LTE to 0.5nM (0.5ml tubes #lib) and fill in the dilution protocol (preferable >2µl aliquots). Total volume: 8.4µl x number of plates

                 c[nM] = 1.5x103 x c[ng/µl] / L[bp]
    c[ng/µl]L[bp]c[nM]



    of nM stock  +   ⇒ 


  2. wash P1 beads:
    • magnetic rack (20 sec) and remove supernatant
    • resuspend in 160µl of BBS
    • vortex (30 sec) and pulse-spin
    • 2 times declumping:
    ○○ Covaris S2: "Bead Block Declump": vortex and pulse-spin in the middle of the program
    ○○ vortex and pulse-spin
    • magnetic rack (1 min) and remove supernatant
    • resuspend in 160µl of TEX
    • vortex (30 sec)
    • pulse-spin and store in ice-box

  3. prepare to set up the ePCR:
    • check that PCR machine is free; check the program's settings
    • prepare XStream pipette, Combitips and wide 200µl tip
    • set 5 min on ULTRA-TURRAX Tube Drive

    NB! §§4-6 should be done non-stop for each PCR plate
  4. add to the aqueous phase (per plate) and store on ice:
    • AmpliTaq Gold, 5u/µl: 600µl
    • template #lib, 0.5nM
    final concentration [pM]volume [µl]
    0.55.6
    0.758.4
    1.011.2
    • vortex beads (30 sec) and pulse-spin
    • 2 times declumping:
    ○○ Covaris S2: "Covalent Declump 1 - beads in 1xTEX": vortex and pulse-spin in the middle of the program
    ○○ vortex and pulse-spin
    • pulse-spin and 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
    • collect aqueous phase with Xtream pipette ("Pip" mode, 10ml Combitip, 1(!) click)
    • insert Combitip in the hole of the TURRAX tube
    • start 5 min run of ULTRA-TURRAX Tube Drive, wait for 5 sec
    • deliver the whole content into the oil phase: first click - small excess; second - main part; and twice more - the rest

    • distribute 150µl aliquots of the emulsion in the wells of PCR plate (should be ~all wells)
    » distribute 3 portions with Xtream pipete ("Dis" mode, 5ml wide Combitip)
    » close the TURRAX tube and shake down the rests of emulsion
    » distribute the rests of emulsion with a wide 200µl tip
    • seal the plate
    • put into PCR machine and start run

  6. after ePCR:
    • check, that emulsion remained stable
    • PCR plate may be stored at +4°C for several days (in the post-PCR area)



[править] Notes

  • stock of P1 beads is ~8x106beads/µl


library

  • stock library should be stored at -20°C at concentration >5ng/µl
  • normal dilution of sequencing library: 0.5nM (~2.5x109 molecules/ePCR plate), which corresponds to 50pg/µl for fragment libraries (~155bp long library molecules) and ~100pg/µl for mate-pair libraries (~300bp long library molecules)


Consumables

ItemOrder number
20ml tube for ePCR water phaseKisker #G111
tubes for ULTRA-TURRAXABI #4400401
96 well plateABI N8010560
Combitip 5mlEppendorf #0030069.250
Combitip 10mlEppendorf #0030069.269


PCR

  • sealing tape: 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)
  • we did not see any differences in the PCR yield or emulsion stability when using MJ research PCR machine or 9700 PCR machine from ABI
  • rough estimate: ePCR has failed if a plate has more than 3-5 wells with broken emulsion. However it is possible to get rid of beads fallen out of the emulsion by pelleting them during bead wash.

  • ePCR program (40 cycles for fragment run; 60 cycles for mate-pair run):
set: "lid tracking with 2°C"
fragment runmate-pair run
[ 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
[ 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, 59 times
[10] 72°C     7:00
[11] 0.5°C to 4°C
[12] 4°C      hold


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