Dinh:Probes Prep May2015

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Revision as of 01:20, 6 May 2015 by >Dinh (→‎PAGE purification (Day 2/3))
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HOTSPOTS 460K[edit]

  • Need more of Subset E. But to save time, I made more of A-E together in case I run out. Noi made one plate of Subsets A-F for me before she left, I still have a lot of subset F to use.

Current probes concentrations[edit]

Subset Qubit (ng/uL)
A 11.84
B 15.4
C 11.92
D 11.76
E ---
F 5.12

Consumed reagents[edit]

  • For future reference to calculate total cost of probes prep for 1.5x96 wells
 6,600 uL KAPA Sybr Fast 2xMM -- $347/10 mL
   900 uL QIAEX II silica beads -- $169/1.5 mL
   180 uL Lambda Exo (5 U/uL)  -- $209/1.0 mL
   210 uL USER (NEB) -- $216/.250 mL
    74 uL DpnII (50 U/uL) -- $211/.100 mL
   12X ssDNA/RNA cleanup & concentrate columns -- $149/50X
  • Total amount above exclude size selection =$ XXX
  • 200 ng gDNA at 200:1 probes to target ratio uses 314.29 ng of probes.
  • Noi's yield is 7-8 ug per plate, Alan's yield is 12-16 ug per plate.
  • If I produce ~6ug of probes, each sample will cost ~ $ XXX in probes (excludes size selection materials).

Probes production (Day 1)[edit]

  • Reagents used: 1.5x96wells plate, 6,600 uL KAPA Sybr Fast 2XMM, 600 uL QIAEX II silica beads
  • Set up ~15 min + 30 min run / 96 reactions = 1.5 hr qPCR
  • PCR cleanup with QIAEXII = 1 hr (would be faster if I can use an ultra-centrifuge to spin down 15 mL tubes).
  • Used 1X2.0 mL tubes to prepare each reaction master mix:
 Reagent                Volume
 KAPA 2X Sybr Fast      1,100 uL
 Nuclease free water    1,100 uL
 AP1V4                  8.8 uL
 AP2V4                  8.8 uL
 Template (10nM)        4.4 uL  (template was prepared by Noi from starting probes mix). 
 95C 30s -> [ 95C 15s -> 55C 45s -> 60C 30s read plate -> 60C 10s]x20cycles -> 60C 1 min 
  • I used the CFX96 to perform qPCR. The incubation time at each temperature was increased to account for the overhead volume (>50uL).
  • Subsets A,C,D appears to saturate at 14 cycles while B,E saturate at 15

File:460K prep May2015 A-E CFX96.jpg

  • I used QIAEX II beads to purify the PCR reactions. Each 2,200 uL total reaction was split into 5x2.0 mL tube.
 Used 20 uL of QIAEX II beads + 3X QX1 buffer.
 Eluted each tube with 50 uL nuclease free water first, then added more water for serial elution.
Subset cycles 100 uL reactions QIAEX II yield, Nanodrop (ng/uL) QIAEX II volume (uL) Total yield (ug)
A 15 22 153.2 220 33.704
B 15 22 128.4 220 28.248
C 15 22 155.5 220 34.21
D 15 22 154 230 35.42
E 14 44 124.6 473 58.9358
  • I expected 33 ug on average from 22 reactions and got about the right amount. Using QIAEX II beads saved time instead of performing ethanol precipitation and Bioneer column purification.

Lambda Exo (Day 1)[edit]

  • Reagents used: 180 uL Lambda Exo (5 U/uL), 300 uL QIAEX II beads.
  • Set up time ~3 hr
Reagent                 Volume
dsDNA+water(~10ug)      125 uL
Lambda Exo buffer,10X   15 uL
Lambda Exo              10 uL
37C 1 hour -> 75C 10 min
  • I set up 3x150 uL reactions for A-D, and 6x150uL reactions for E. I expected <20ug after digestion, so after pooling 3 reactions together in a 2.0 mL tube, I purified with 50 uL QIAEX II beads and 3X volumes QX1 buffer.
  • Yield:
Subset Input(ug) QIAEX II yield, Nanodrop (ng/uL) QIAEX II volume (uL) Total LambdaExo/QIAEXII yield (ug) %yield
A 33.704 76.5 190 14.535 43%
B 28.248 62.5 190 11.875 42%
C 34.21 79.2 190 15.048 44%
D 35.42 81.5 190 15.485 44%
E 29.4679 67.1 190 12.749 43%
E 29.4679 69.1 190 13.129 45%
  • Check Lambda Exo efficiency by running PAGE TBE gel.
    • LambdaExo digestion have good efficiency.
    • QIAEX II can purify ssDNA

File:460K prep May2015 A-E LambdaExoDigestion.jpg

USER digestion (Day 1/Overnight)[edit]

  • Reagents used: 210 uL USER (NEB)
  • Set up time ~ 1 hr
  • Set up 1x1.5 mL tube for each overnight reactions.
 Reagent                       Volume
 ssDNA+water(up to 15 ug)      280 uL
 DpnII buffer (10X)            35 uL
 USER (NEB)                    35 uL
 37C for overnight in thermomixer, 400 rpm
  • Aliquot 2 uL of each (A-E) for PAGE-verification.

DpnII digestion (Day 2)[edit]

  • Reagents used: 62 uL DpnII (50 U/uL)
  • Set up time 30 minutes + 2 hours incubation + 30 minutes cleanup = 3 hr.
  • To each USER 350 uL reaction, add 40 uL Guide-oligo (NN version), and add 5 uL DpnII buffer = 395 uL. Split into 4x0.2mL PCR tubes (~100 uL each).
 94C for 2 minutes -> 0.1C/s to 60C -> 60C for 20 minutes -> 37C for 3 minutes add DpnII
 To each PCR tube, add a total of 5 uL of DpnII to each set (50U/uL).
 Use P200 to mix each reaction by re-pipetting 10 times on the thermocycler.
  • Incubate at 37C for 1 hour.
  • Aliquot 2 uL of each (A-E) for PAGE-verification
  • After 1 hour, add mixture of 8 uL water, 1 uL DpnII buffer, 1 uL of DpnII
    • NOTE: I forgot to ramp up to 94C and then back to 37C. This would have helped the digestion efficiency more than simply adding more DpnII.
 Reagent                  Volume
 Nuclease free water      8x31 = 248 uL
 DpnII buffer (10X)       1x31 = 31 uL
 DpnII                    1x31 = 31 uL
 10 uL of DpnII mixture to each, use P200 to mix each reaction by repipetting 10 times on the thermocycler.
  • Incubate at 37C for 1 more hour.
  • Aliquot 2 uL of each (A-E) for PAGE-verification
  • Clean up 5x90 uL reactions using 2 ssDNA cleanup columns (Zymo). Use ssDNA column to remove leftover dsDNA!
  • Yield:
Subset Input(ug) USER/DpnII, ssDNA column, QUBIT(ng/uL) ssDNA elution volume Total (ug) %yield
A 14.535 27.4 115 3.151 22%
B 11.875 21.6 115 2.484 21%
C 15.048 25.2 115 2.898 19%
D 15.485 33.6 115 3.864 25%
E 12.749 20.6 115 2.369 19%
F 13.129 25.2 115 2.898 22%
  • Gel confirmed that DpnII digestion was incomplete, use 100 units and RE_DpnII_V4 (no Ns) to try removing more adaptors.
  • Gel after USER (O/N), DpnII (1hr), DpnII (1hr)
 File:460k A-E prep USER DpnII DpnII ABC.jpg
 File:460k A-E prep USER DpnII DpnII DEE.jpg
Subset Total (ug) ssDNA elution volume RE_DpnII_V4 oligo (uL) DpnII (uL) DpnII buffer(10x) Water(uL)
A 3.1784 116 15 2 15 4
B 2.5272 117 15 2 15 3
C 2.9484 117 15 2 15 3
D 3.9312 117 15 2 15 3
E 2.4102 117 15 2 15 3
E 2.9484 117 15 2 15 3
  • Gel after final digestion:
 File:460k A-E prep USER DpnII DpnII DpnII ABCDEE.jpg

PAGE purification (Day 2/3)[edit]

  • Purified A, B
  • Reagent: 4 TBE-Urea gels, 300 uL TBU loading buffer, 6 Nanosep columns.
  • We ran out of agarose so I used PAGE instead.
  • Prepared 2D TBE-Urea gels for loading. Loaded 75 uL of sample + 75 uL of 2x loading buffer into each well. I used 2 gels per sample (22 PCR reactions).
  • I didn't use a ladder because I already ran these samples with a ladder on the digestion check gel.
  • Prepared the gel-shearing apparatus (1x2.0 mL tube per 2D gel with 1x0.5 mL inside - pierced the 0.5 mL tube with a 20 gauge needle, cut off the cap of the 2.0 mL tube and save for later) and UV them.
  • Ran the gels at 220 V for 30 minutes. Stain with Sybr Gold for ~30 seconds.
  • Wrap each gel flat with plastic wrap and under UV light, use a Sharpie marker to trace the band to be excised on the plastic wrap.
  • Use a No. 10 scapel to cut out the desired bands and transfer sliced gel into the pierced 0.5 mL tube. Image the gel within the plastic wrap to ensure correct fragment was excised.
  • Spin the tubes at maximum speed for 2 minutes.
  • To each tube, at 3X(gel volume) of 1X TE buffer. I used 1.4 mL of TE per gel. Cap & freeze the tubes at -80C
  • Thaw each tube in the thermomixer for 30 minutes at 50C. Ramp temperature up to 94C and soak for 5 minutes. Ramp temperature down to 37C and soak for 30 minutes.
  • Separate gel from elutant using 1 Nanosep column (3 rounds centrifugation) per tube of gel. Transfer the elutant to a 5.0 mL tube.
  • To each 5.0 mL tube with 1.4 mL elutant, add 140 uL 3.0 M sodium acetate, 5 uL GlycoBlue, 3.5 mL of 100% ethanol. Cap & freeze each tube at -80C.
  • Use the ultracentrifuge in the basement to (SORVALL) to spin down each 5.0 mL tubes at 12,000rpm for 20 minutes.
  • Transfer the pellet to a 1.5 mL tube with ice cold 75% ethanol & spin down & dry. After drying dissolve each pellet with 50 uL of water.
Subset Total QUBIT (ug) PAGE/EtohPPT (ng/uL) Volume (uL) Yield QUBIT (ug) %yield
A 3.1784 8.12 50 0.406 12.77%
B 2.5272 7.72 50 0.386 15.27%