Noi/NOTES/2011-9-18

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BSPP DMR330k capture of samples from Sergio, two largest pedigrees from HAPMAPPT01, ten replicates, HFF somatic lines, repeat UPenn samples, and comparing DNA methylation kit from Zymoresearch and Sigma[edit]

  • Continued from 2011_09_16: [[1]]

Capture set up[edit]

Subset ID Adjusted Multiplier based on qPCR (Multiplier E) Actual amount of probe required (probe:target ratio, 100:1) Multiplier E x amount probe required Probe concentration (ng/ul) Volume (ul) Volume for 55rxn (ul) '
DMR.s1 0.05 2.24 0.11 1.61 0.07 3.83 DMR.s1
DMR.s2 0.07 2.24 0.16 3.16 0.05 2.83 DMR.s2
DMR.s3 0.23 18.90 4.41 21.15 0.21 11.46 DMR.s3
DMR.s4 1.00 18.90 18.90 22.34 0.85 46.53 DMR.s4
DMR.s5 4.83 18.90 91.29 22.14 4.12 226.77 DMR.s5
DMR.Exp1 0.23 18.90 4.44 31.88 0.14 7.65 DMR.Exp1
DMR.Exp2 0.19 18.90 3.50 24.11 0.15 8.00 DMR.Exp2
DMR.Exp3 0.27 18.90 5.03 28.90 0.17 9.56 DMR.Exp3
1.05uM Oligo suppressor-set1 0.15 8.25 1.05uM Oligo suppressor-set1
1.05uM Oligo suppressor-set2 0.15 8.25 1.05uM Oligo suppressor-set2
Total volume only probe 5.76


Reaction mix[edit]

Components 1rxn (ul) 55.00
10X AmpLigase Buffer 2.60 143.00
DMR330k probe mix (ul) 5.76 316.80
105nM Oligo suppressor 0.30 16.50
Total volume (ul) 8.66 476.30
  • Aliquot 59.5ul in strip tube
Sample ID Final conc. (ng/ul) Volume for 250ng(ul) Well ID sample ID H2O (ul) Total amount of bis-cvt gDNA (ng) Probe mix (ul) 10x Amligase buffer (ul) 1.05uM Oligo suppressor set1&2 (ul) Total volume (ul)
1. H9 15.28 16.36 A1 1. H9 0.98 250.00 5.76 2.60 0.30 26.00
**2. H9+Activin 100ng/mL 5days NA NA B1 **2. H9+Activin 100ng/mL 5days NA NA NA 2.60 NA NA
3. H9+BMP4 50ng/mL 5days 22.00 11.36 C1 3. H9+BMP4 50ng/mL 5days 5.98 250.00 5.76 2.60 0.30 26.00
4. HUViPS4F1 23.40 10.68 D1 4. HUViPS4F1 6.66 250.00 5.76 2.60 0.30 26.00
5. HUViPS4F1+Activin 100ng/mL 5days 15.38 16.25 E1 5. HUViPS4F1+Activin 100ng/mL 5days 1.09 250.00 5.76 2.60 0.30 26.00
6. HUViPS4F1+BMP4 50ng/mL 5days 22.80 10.96 F1 6. HUViPS4F1+BMP4 50ng/mL 5days 6.38 250.00 5.76 2.60 0.30 26.00
7. HUViPS4F3 23.60 10.59 G1 7. HUViPS4F3 6.75 250.00 5.76 2.60 0.30 26.00
8. HUViPS4F3+Activin 100ng/mL 5days 15.86 15.76 H1 8. HUViPS4F3+Activin 100ng/mL 5days 1.58 250.00 5.76 2.60 0.30 26.00
9. HUViPS4F3+BMP4 50ng/mL 5days 19.78 12.64 A3 9. HUViPS4F3+BMP4 50ng/mL 5days 4.70 250.00 5.76 2.60 0.30 26.00
GM11995 (wellID G7) 41.00 6.10 B3 GM11995 (wellID G7) 11.24 250.00 5.76 2.60 0.30 26.00
GM11993 (wellID F2) 16.16 15.47 C3 GM11993 (wellID F2) 1.87 250.00 5.76 2.60 0.30 26.00
**GM10861 (wellID A9) 10.36 NA D3 **GM10861 (wellID A9) NA NA NA NA NA NA
GM11992 (wellID C2 ) 22.40 11.16 E3 GM11992 (wellID C2 ) 6.18 250.00 5.76 2.60 0.30 26.00
GM11994 (wellID G9 ) 27.60 9.06 F3 GM11994 (wellID G9 ) 8.28 250.00 5.76 2.60 0.30 26.00
GM10860 (wellID F10) 23.20 10.78 G3 GM10860 (wellID F10) 6.56 250.00 5.76 2.60 0.30 26.00
GM12813 (wellID E8) 35.20 7.10 H3 GM12813 (wellID E8) 10.24 250.00 5.76 2.60 0.30 26.00
GM12802 (wellID E7) 26.60 9.40 A5 GM12802 (wellID E7) 7.94 250.00 5.76 2.60 0.30 26.00
GM12815 (wellID G12) 28.00 8.93 B5 GM12815 (wellID G12) 8.41 250.00 5.76 2.60 0.30 26.00
GM12812 (wellID F11) 22.40 11.16 C5 GM12812 (wellID F11) 6.18 250.00 5.76 2.60 0.30 26.00
GM12814 (wellID A7) 19.42 12.87 D5 GM12814 (wellID A7) 4.47 250.00 5.76 2.60 0.30 26.00
GM12801 (wellID D9) 24.40 10.25 E5 GM12801 (wellID D9) 7.09 250.00 5.76 2.60 0.30 26.00
NA12156 (wellID B3)-1 15.05 16.61 F5 NA12156 (wellID B3)-1 0.73 250.00 5.76 2.60 0.30 26.00
NA12156 (wellID B3)-2 15.05 16.61 G5 NA12156 (wellID B3)-2 0.73 250.00 5.76 2.60 0.30 26.00
NA12156 (wellID B3)-3 15.05 16.61 H5 NA12156 (wellID B3)-3 0.73 250.00 5.76 2.60 0.30 26.00
NA12156 (wellID B3)-4 15.05 16.61 A7 NA12156 (wellID B3)-4 0.73 250.00 5.76 2.60 0.30 26.00
NA12156 (wellID B3)-5 15.05 16.61 B7 NA12156 (wellID B3)-5 0.73 250.00 5.76 2.60 0.30 26.00
NA12156 (wellID B3)-6 15.05 16.61 C7 NA12156 (wellID B3)-6 0.73 250.00 5.76 2.60 0.30 26.00
NA12156 (wellID B3)-7 15.05 16.61 D7 NA12156 (wellID B3)-7 0.73 250.00 5.76 2.60 0.30 26.00
NA12156 (wellID B3)-8 15.05 16.61 E7 NA12156 (wellID B3)-8 0.73 250.00 5.76 2.60 0.30 26.00
NA12156 (wellID B3)-9 15.05 16.61 F7 NA12156 (wellID B3)-9 0.73 250.00 5.76 2.60 0.30 26.00
NA12156 (wellID B3)-10 15.05 16.61 G7 NA12156 (wellID B3)-10 0.73 250.00 5.76 2.60 0.30 26.00
**2HFF P6 8.80 NA H7 **2HFF P6 NA NA NA NA NA NA
**218 HFF P7 8.24 NA A9 **218 HFF P7 NA NA NA NA NA NA
KEBR028A (B4-plate) 7.70 17.00 B9 KEBR028A (B4-plate) 0.34 130.90 5.76 2.60 0.30 26.00
NTC 0.00 0.00 C9 NTC 17.34 0.00 5.76 2.60 0.30 26.00
KEBR040 (D4-plate) 31.40 12.74 D9 KEBR040 (D4-plate) 4.60 400.00 5.76 2.60 0.30 26.00
TZHZ018 (F1-plate) 27.40 14.60 E9 TZHZ018 (F1-plate) 2.74 400.00 5.76 2.60 0.30 26.00
TZHZ031A (tube) 54.80 7.30 F9 TZHZ031A (tube) 10.04 400.00 5.76 2.60 0.30 26.00
TZHZ041A (tube) 43.00 9.30 G9 TZHZ041A (tube) 8.04 400.00 5.76 2.60 0.30 26.00
TZHZ075 (H01-plate) 32.80 12.20 H9 TZHZ075 (H01-plate2) 5.14 400.00 5.76 2.60 0.30 26.00
**PGP1iPS 6.00 NA A11 **PGP1iPS NA NA NA NA NA NA
PGP1iPS-IMPRINT 8.58 17.00 B11 PGP1iPS-IMPRINT 0.34 145.86 5.76 2.60 0.30 26.00
**Hues1 7.16 34.92 C11 **Hues1 NA NA NA NA NA NA
**Hues3 6.92 36.13 D11 **Hues3 NA NA NA NA NA NA
**Hues8 8.68 28.80 E11 **Hues8 NA NA NA NA NA NA


Sample ID Final conc. (ng/ul) Volume for 250ng(ul) Well ID sample ID H2O (ul) Total amount of bis-cvt gDNA (ng) Probe mix (ul) 10x Amligase buffer (ul) 1.05uM Oligo suppressor set1&2 (ul) Total volume (ul)
2. H9+Activin 100ng/mL 5days 14.50 17.24 B1 2. H9+Activin 100ng/mL 5days 0.10 250.00 5.76 2.60 0.30 26.00
GM10861 (wellID A9) 14.80 16.89 D3 GM10861 (wellID A9) 0.45 250.00 5.76 2.60 0.30 26.00
2HFF P6 18.40 13.59 H7 2HFF P6 3.75 250.00 5.76 2.60 0.30 26.00
218 HFF P7 18.00 13.89 A9 218 HFF P7 3.45 250.00 5.76 2.60 0.30 26.00
PGP1iPS 9.08 16.08 A11 PGP1iPS 1.26 146.00 5.76 2.60 0.30 26.00
PGP1F 16.50 15.15 F11 PGP1F 2.19 250.00 5.76 2.60 0.30 26.00
Hues1 13.20 18.94 C11 Hues1 0.00 250.00 5.76 2.60 0.30 27.60
Hues3 13.30 18.80 D11 Hues3 0.00 250.00 5.76 2.60 0.30 27.46
Hues8 15.30 16.34 E11 Hues8 1.00 250.00 5.76 2.60 0.30 26.00


  • Add H2O to the 96-well plate first then add bis-cvt gDNA. Note: Try to deliver reagents to the bottom of the well
  • Prepare reaction mix in 1.5 mL tube by pipetting probe, oligo suppressor and 10x AmLigase buffer and mix well.
  • Aliquot reaction mix in striptube (8 tubes): 59.5ul
  • Aliquot reaction mix 8.66ul with multichannel pipette and mix well
  • Put mineral oil in basin ~2 mL
  • Layer the capture reaction with 25ul mineral oil with multichannel pipette.
  • Seal the plate with microseal and make sure that every well is completely sealed to prevent evaporation
  • Check the level of every well that they have the same level
  • Centrifuge the capture plate for 3 min and place the plate on PCR machine with the following profile,

Program

-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h 
-> add 2.5ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP) (2011_09_20-->12:30am)
-> 55C 13h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. (2011_09_20)

SLN mix[edit]

Components Stock conc. Unit Final conc. Unit Prepare volume 138ul
AmpliTaq Stoffel 10.00 U/ul 2.00 U/ul 27.60
AmpLigase 5.00 U/ul 0.50 U/ul 13.80
dNTP 1000.00 uM 100.00 uM 13.80
10x AmpLigase Buffer 10.00 x 1.00 x 13.80
H2O 69.00
Total 138.00
  • Aliquot 17.25 ul to stripp tube
  • Add 2.5ul of SLN mix to each well with multichannel pipettes

ExoI/III preparation[edit]

  • Mix 55 ul each of ExoI and ExoIII and aliquot 13.75ul into striptube
  • Add 2ul to each well with multichannel pipettes

PCR Amplification with barcoded primers (AmpF6.4Sol and AmpR6.3 IndX No.1-46)[edit]

  • Spin the capture plate for 3 min to collect all liquid to the bottom of the well
  • Test sample H5 and H7 to see if the capture work
Components 1 rxn 2.2 rxn
Captured template 2.00 0.00
10uM AmpF6.4Sol 1.00 2.20
10uM AmpR6.3 Sol 1.00 2.20
50x SYBG 0.40 0.88
2x Phusion MM 25.00 55.00
H2O 20.60 45.32
Total 50.00 110.00
  • Aliquot 48ul, add 2ul of captured DNA

Program 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min

Components 1rxn 50 rxn
Captured template 10.00 0.00
10uM AmpF6.4Sol 2.00 100.00
10uM AmpR6.3 (IndX, X=1-46) 2.00 0.00
50x SYBG 0.80 40.00
2x Phusion MM 50.00 2500.00
H2O 35.20 1760.00
Total 100.00 5000.00
  • aliquot 88ul, add 2ul AmpR6.3Ind primer and 10ul DNA template

Program 98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min

A1 A3 A5 A7 A9 A11
AmpR6.3.Ind1 AmpR6.3.Ind9 AmpR6.3.Ind17 AmpR6.3.Ind25 AmpR6.3.Ind33 AmpR6.3.Ind41
B1 B3 B5 B7 B9 B11
AmpR6.3.Ind2 AmpR6.3.Ind10 AmpR6.3.Ind18 AmpR6.3.Ind26 AmpR6.3.Ind34 AmpR6.3.Ind42
C1 C3 C5 C7 C9 C11
AmpR6.3.Ind3 AmpR6.3.Ind11 AmpR6.3.Ind19 AmpR6.3.Ind27 AmpR6.3.Ind35 AmpR6.3.Ind43
D1 D3 D5 D7 D9 D11
AmpR6.3.Ind4 AmpR6.3.Ind12 AmpR6.3.Ind20 AmpR6.3.Ind28 AmpR6.3.Ind36 AmpR6.3.Ind44
E1 E3 E5 E7 E9 E11
AmpR6.3.Ind5 AmpR6.3.Ind13 AmpR6.3.Ind21 AmpR6.3.Ind29 AmpR6.3.Ind37 AmpR6.3.Ind45
F1 F3 F5 F7 F9 F11
AmpR6.3.Ind6 AmpR6.3.Ind14 AmpR6.3.Ind22 AmpR6.3.Ind30 AmpR6.3.Ind38 AmpR6.3.Ind46
G1 G3 G5 G7 G9
AmpR6.3.Ind7 AmpR6.3.Ind15 AmpR6.3.Ind23 AmpR6.3.Ind31 AmpR6.3.Ind39
H1 H3 H5 H7 H9
AmpR6.3.Ind8 AmpR6.3.Ind16 AmpR6.3.Ind24 AmpR6.3.Ind32 AmpR6.3.Ind40
Plate layout 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00
A Sergio1 Sergio9 GM12802 (wellID E7) NA12156 (wellID B3)-4 218 HFF P7 PGP1iPS
B Sergio2 GM11995 (wellID G7) GM12815 (wellID G12) NA12156 (wellID B3)-5 KEBR028A (B4-plate) PGP1iPS (Imprint)
C Sergio3 GM11993 (wellID F2) GM12812 (wellID F11) NA12156 (wellID B3)-6 NTC Hues1
D Sergio4 GM10861 (wellID A9) GM12814 (wellID A7) NA12156 (wellID B3)-7 KEBR040 (D4-plate) Hues3
E Sergio5 GM11992 (wellID C2 ) GM12801 (wellID D9) NA12156 (wellID B3)-8 TZHZ018 (F1-plate) Hues8
F Sergio6 GM11994 (wellID G9 ) NA12156 (wellID B3)-1 NA12156 (wellID B3)-9 TZHZ031A (tube) PGP1F
G Sergio7 GM10860 (wellID F10) NA12156 (wellID B3)-2 NA TZHZ041A (tube)
H Sergio8 GM12813 (wellID E8) NA12156 (wellID B3)-3 2HFF P6 TZHZ075 (H01-plate)
Well ID conc. (ng/ul) Total amount in 60ul (ng) Volume for 60ng (ul) Well ID
A1 4.18 250.84 14.35 A1
B1 1.18 70.83 50.82 B1
C1 1.99 119.37 30.16 C1
D1 1.79 107.11 33.61 D1
E1 2.69 161.57 22.28 E1
F1 4.30 258.01 11.63 F1
G1 1.83 109.97 32.74 G1
H1 5.53 331.90 10.85 H1
A3 2.91 174.72 20.60 A3
B3 4.63 277.76 10.80 B3
C3 4.51 270.42 11.09 C3
D3 4.36 261.87 11.46 D3
E3 4.85 290.73 10.32 E3
F3 2.27 136.34 22.00 F3
G3 4.55 273.09 10.99 G3
H3 7.16 429.67 6.98 H3
A5 6.35 380.88 7.88 A5
B5 6.68 401.08 7.48 B5
C5 4.90 294.04 10.20 C5
D5 7.65 459.30 6.53 D5
E5 7.06 423.81 7.08 E5
F5 6.73 403.67 7.43 F5
G5 7.75 465.10 6.45 G5
H5 7.70 461.92 6.49 H5
A7 5.87 352.50 8.51 A7
B7 4.94 296.31 10.12 B7
C7 6.67 400.41 7.49 C7
D7 6.56 393.87 7.62 D7
E7 5.05 302.89 9.90 E7
F7 6.77 406.00 7.39 F7
H7 5.86 351.46 8.54 H7
A9 3.26 195.75 15.33 A9
B9 NA NA NA B9
C9 (NTC) NA NA NA C9 (NTC)
D9 1.89 113.62 26.40 D9
E9 NA NA NA E9
F9 5.29 317.33 9.45 F9
G9 5.30 317.86 9.44 G9
H9 4.72 283.20 10.59 H9
A11 4.24 254.15 14.16 A11
B11 3.44 206.65 17.42 B11
C11 2.90 173.82 20.71 C11
D11 3.24 194.55 18.50 D11
E11 4.00 239.91 15.01 E11
UCLA-RO-IND94 12.31 369.16 4.06 UCLA-RO-IND94


  • Note: Need to fill mor einfo ....
  • Sequencing ID: NP-BSPP-Ind1-45_Sept18
  • HiSeq run HL104 at Cancer Center
  • Continued on: 2011-10-17 (after HL104 Hiseq run): [[2]]