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]]