Noi/NOTES/2010-12-18
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DMR220k capture and library construction of normal/tumor samples from JHU[edit]
continued from 2010_12_16: [[1]]
Capture set up[edit]
Probe/Target Ratio | 100 |
Probes size | 220,000 |
Template amount | 200ng |
Probe calculation[edit]
- use multiplier C for probe calculation. this table for 200ng probes
Subset ID | Multiplier C | Amount probes required x multiplier (ng) | Conc(ng/ul) | Volume (ul) | 53rxn mix |
DMR.s1 | 0.05 | 0.11 | 2.00 | 0.06 | 2.92 |
DMR.s2 | 0.09 | 0.21 | 2.00 | 0.11 | 5.57 |
DMR.s3 | 0.25 | 4.71 | 23.20 | 0.20 | 10.76 |
DMR.s4 | 1.00 | 18.88 | 24.80 | 0.76 | 40.35 |
DMR.s5 | 4.83 | 91.22 | 26.70 | 3.42 | 181.07 |
DMR.s6 | 4.83 | 44.78 | 27.70 | 1.62 | 85.68 |
DMR.s7 | 4.83 | 44.78 | 32.00 | 1.40 | 74.17 |
Total volume (ul) | 7.56 | 400.51 |
Reaction mix[edit]
Components | 1rxn (ul) | 53rxn mix (ul) |
10x AmpLigase buffer (ul) | 2.50 | 132.50 |
DMR220k probe mix (ul) | 7.56 | 400.68 |
105nM oligo suppressor (ul) | 1.00 | 53.00 |
Total vol. (ul) | 11.06 | 586.18 |
- aliquot approx 73ul of rxn mix to strip tubes (8 tubes)
- pipette 11.06ul of rxn mix from strip tube to 96-well plate containing BIS-CVT gDNA with multi-channel pipette
- mix by pipetting, add 20ul of mineral oil
- seal the plate with microseal film
- spin capture plate down
Samples | Concentration (ng/ul) | Volume (ul) for appox. 200ng | H2O(ul) | 10x AmpLigase buffer (ul) | DMR220k probe mix (ul) | 105nM oligo suppressor | Total volume |
A1 | 41.60 | 5.00 | 8.94 | 2.50 | 7.56 | 1.00 | 25.00 |
B1 | 20.20 | 10.00 | 3.94 | 2.50 | 7.56 | 1.00 | 25.00 |
C1 | 27.00 | 7.50 | 6.44 | 2.50 | 7.56 | 1.00 | 25.00 |
D1 | 1.61 | 14.00 | 0.00 | 2.50 | 7.56 | 1.00 | 25.06 |
E1 | 25.60 | 8.00 | 5.94 | 2.50 | 7.56 | 1.00 | 25.00 |
F1 | 24.60 | 8.20 | 5.74 | 2.50 | 7.56 | 1.00 | 25.00 |
G1 | 25.80 | 7.80 | 6.14 | 2.50 | 7.56 | 1.00 | 25.00 |
H1 | 31.20 | 6.50 | 7.44 | 2.50 | 7.56 | 1.00 | 25.00 |
A2 | 13.86 | 14.00 | 0.00 | 2.50 | 7.56 | 1.00 | 25.06 |
B2 | 29.00 | 7.00 | 6.94 | 2.50 | 7.56 | 1.00 | 25.00 |
C2 | 27.00 | 7.50 | 6.44 | 2.50 | 7.56 | 1.00 | 25.00 |
D2 | 20.40 | 10.00 | 3.94 | 2.50 | 7.56 | 1.00 | 25.00 |
E2 | 15.68 | 13.00 | 0.94 | 2.50 | 7.56 | 1.00 | 25.00 |
F2 | 13.80 | 14.00 | 0.00 | 2.50 | 7.56 | 1.00 | 25.06 |
G2 | 26.00 | 7.70 | 6.24 | 2.50 | 7.56 | 1.00 | 25.00 |
H2 | 24.80 | 8.10 | 5.84 | 2.50 | 7.56 | 1.00 | 25.00 |
A3 | 21.00 | 9.50 | 4.44 | 2.50 | 7.56 | 1.00 | 25.00 |
B3 | 72.40 | 2.80 | 11.14 | 2.50 | 7.56 | 1.00 | 25.00 |
C3 | 27.00 | 7.50 | 6.44 | 2.50 | 7.56 | 1.00 | 25.00 |
D3 | 24.60 | 8.20 | 5.74 | 2.50 | 7.56 | 1.00 | 25.00 |
E3 | 48.60 | 4.20 | 9.74 | 2.50 | 7.56 | 1.00 | 25.00 |
F3 | 30.40 | 6.60 | 7.34 | 2.50 | 7.56 | 1.00 | 25.00 |
G3 | 31.40 | 6.50 | 7.44 | 2.50 | 7.56 | 1.00 | 25.00 |
H3 | 22.00 | 9.10 | 4.84 | 2.50 | 7.56 | 1.00 | 25.00 |
A4 | 27.00 | 7.50 | 6.44 | 2.50 | 7.56 | 1.00 | 25.00 |
B4 | 29.80 | 6.80 | 7.14 | 2.50 | 7.56 | 1.00 | 25.00 |
C4 | 13.48 | 14.00 | 0.00 | 2.50 | 7.56 | 1.00 | 25.06 |
D4 | 28.00 | 7.20 | 6.74 | 2.50 | 7.56 | 1.00 | 25.00 |
E4 | 24.60 | 8.20 | 5.74 | 2.50 | 7.56 | 1.00 | 25.00 |
F4 | 9.16 | 14.00 | 0.00 | 2.50 | 7.56 | 1.00 | 25.06 |
G4 | 20.80 | 9.70 | 4.24 | 2.50 | 7.56 | 1.00 | 25.00 |
H4 | 10.32 | 14.00 | 0.00 | 2.50 | 7.56 | 1.00 | 25.06 |
A5 | 24.00 | 8.40 | 5.54 | 2.50 | 7.56 | 1.00 | 25.00 |
B5 | 14.48 | 13.80 | 0.14 | 2.50 | 7.56 | 1.00 | 25.00 |
C5 | 9.92 | 14.00 | 0.00 | 2.50 | 7.56 | 1.00 | 25.06 |
D5 | 25.40 | 8.00 | 5.94 | 2.50 | 7.56 | 1.00 | 25.00 |
E5 | 24.40 | 8.20 | 5.74 | 2.50 | 7.56 | 1.00 | 25.00 |
F5 | 32.00 | 6.25 | 7.69 | 2.50 | 7.56 | 1.00 | 25.00 |
G5 | 25.20 | 8.00 | 5.94 | 2.50 | 7.56 | 1.00 | 25.00 |
H5 | 29.00 | 7.00 | 6.94 | 2.50 | 7.56 | 1.00 | 25.00 |
A6 | 0.20 | 14.00 | 0.00 | 2.50 | 7.56 | 1.00 | 25.06 |
B6 | 26.00 | 7.70 | 6.24 | 2.50 | 7.56 | 1.00 | 25.00 |
C6 | 104.40 | 2.00 | 11.94 | 2.50 | 7.56 | 1.00 | 25.00 |
D6 | 53.60 | 3.80 | 10.14 | 2.50 | 7.56 | 1.00 | 25.00 |
E6 | 31.00 | 6.50 | 7.44 | 2.50 | 7.56 | 1.00 | 25.00 |
F6 | 38.20 | 5.30 | 8.64 | 2.50 | 7.56 | 1.00 | 25.00 |
G6 | 34.60 | 5.80 | 8.14 | 2.50 | 7.56 | 1.00 | 25.00 |
H6 | 30.80 | 6.50 | 7.44 | 2.50 | 7.56 | 1.00 | 25.00 |
NTC | 0.00 | 0.00 | 13.94 | 2.50 | 7.56 | 1.00 | 25.00 |
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 58C 20h (2010_12_18) -> add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 50uM dNTP) (2010_12_19) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. (2010_12_20)
SLN mix preparation[edit]
Components | Stock conc. | Final conc. | 55 rxn mix |
AmpliTaq Stoffel | 10U/ul | 2.00 | 22.00 |
AmpLigase | 5U/ul | 0.50 | 11.00 |
dNTP | 1000uM or 1mM | 50uM | 5.50 |
10x AmpLigase Buffer | 10x | x | 11.00 |
H2O | 60.50 | ||
Total volume (ul) | 110.00 |
- aliquot 13.75ul of SLN mix into strip tubes
- add 2ul to each well with multi-channel pipette
- Note: the SLN mix was not enough, next time about 60rxn should be prepared for 49 rxn.
2010_12_21
Amplification[edit]
total samples 48+1NTC
total volume of captured DNA 29ul
Components | 1x | 51 rxn mix |
Template (captured DNA) | 10 | 0 |
10uM AmpF6.3Sol | 2 | 102 |
10uMAmpR6.3IndX(X=1,2,3,…,48) | 2 | 0 |
50x SYBG | 0.4 | 20.4 |
2x Phusion MM | 50 | 2550 |
H2O | 35.6 | 1815.6 |
total | 100 | aliquot 88ul, add 2ul AmpR6.3IndX, 10ul template |
Samples | Ind | Samples | Ind | Samples | Ind | Samples | Ind | Samples | Ind | Samples | Ind |
A1 | R6.3 IndX1 | A2 | R6.3 IndX9 | A3 | R6.3 IndX17 | A4 | R6.3 IndX25 | A5 | R6.3 IndX33 | A6 | R6.3 IndX41 |
B1 | R6.3 IndX2 | B2 | R6.3 IndX10 | B3 | R6.3 IndX18 | B4 | R6.3 IndX26 | B5 | R6.3 IndX34 | B6 | R6.3 IndX42 |
C1 | R6.3 IndX3 | C2 | R6.3 IndX11 | C3 | R6.3 IndX19 | C4 | R6.3 IndX27 | C5 | R6.3 IndX35 | C6 | R6.3 IndX43 |
D1 | R6.3 IndX4 | D2 | R6.3 IndX12 | D3 | R6.3 IndX20 | D4 | R6.3 IndX28 | D5 | R6.3 IndX36 | D6 | R6.3 IndX44 |
E1 | R6.3 IndX5 | E2 | R6.3 IndX13 | E3 | R6.3 IndX21 | E4 | R6.3 IndX29 | E5 | R6.3 IndX37 | E6 | R6.3 IndX45 |
F1 | R6.3 IndX6 | F2 | R6.3 IndX14 | F3 | R6.3 IndX22 | F4 | R6.3 IndX30 | F5 | R6.3 IndX38 | F6 | R6.3 IndX46 |
G1 | R6.3 IndX7 | G2 | R6.3 IndX15 | G3 | R6.3 IndX23 | G4 | R6.3 IndX31 | G5 | R6.3 IndX39 | G6 | R6.3 IndX47 |
H1 | R6.3 IndX8 | H2 | R6.3 IndX16 | H3 | R6.3 IndX24 | H4 | R6.3 IndX32 | H5 | R6.3 IndX40 | H6 | R6.3 IndX48 |
Program
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x15 -> 72C 3min
(2010_12_22)
Amplification[edit]
- total samples (48 x 2 = 96) no NTC
Components | 1x | 100 rxn mix |
Template (captured DNA) | 9 | 0 |
10uM AmpF6.3Sol | 2 | 200 |
10uMAmpR6.3IndX(X=1,2,3,…,48) | 2 | 0 |
50x SYBG | 0.4 | 40 |
2x Phusion MM | 50 | 5000 |
H2O | 36.6 | 3660 |
total volume (ul) | 100 | aliquot 89ul, add 2ul AmpR6.3IndX, 9ul template |
Plate layout
Well No. | ///1/// | ///2/// | ///3/// | ///4/// | ///5/// | ///6/// | ///7/// | ///8/// | ///9/// | ///10/// | ///11/// | ///12/// |
A | A1 | A1 | A2 | A2 | A3 | A3 | A4 | A4 | A5 | A5 | A6 | A6 |
B | B1 | B1 | B2 | B2 | B3 | B3 | B4 | B4 | B5 | B5 | B6 | B6 |
C | C1 | C1 | C2 | C2 | C3 | C3 | C4 | C4 | C5 | C5 | C6 | C6 |
D | D1 | D1 | D2 | D2 | D3 | D3 | D4 | D4 | D5 | D5 | D6 | D6 |
E | E1 | E1 | E2 | E2 | E3 | E3 | E4 | E4 | E5 | E5 | E6 | E6 |
F | F1 | F1 | F2 | F2 | F3 | F3 | F4 | F4 | F5 | F5 | F6 | F6 |
G | G1 | G1 | G2 | G2 | G3 | G3 | G4 | G4 | G5 | G5 | G6 | G6 |
H | H1 | H1 | H2 | H2 | H3 | H3 | H4 | H4 | H5 | H5 | H6 | H6 |
Label | 1 | 1 | 2 | 2 | 3 | 3 | 4 | 4 | 5 | 5 | 6 | 6 |
Program
98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x12 -> 72C 3min
File:2010 12 21 LibConAmpFR63sol.png
- 25ng from each library was combined and PAGE size selection with 6% TBE 5-well PAGE gel (4 lanes)
Samples | Final conc.(ng/ul) | Yields (ng) in 40ul | Volume for 25ng (ul) |
A1 | 7.46 | 298.40 | 3.35 |
E1 | 6.91 | 276.51 | 3.62 |
B1 | 6.50 | 259.91 | 3.85 |
F1 | 7.62 | 304.66 | 3.28 |
C1 | 6.63 | 265.34 | 3.77 |
G1 | 9.15 | 366.16 | 2.73 |
D1* | 0.58 | 23.36 | 20* |
H1 | 5.39 | 215.58 | 4.64 |
A2 | 5.29 | 211.68 | 4.72 |
E2 | 6.10 | 244.14 | 4.10 |
B2 | 3.51 | 140.51 | 7.12 |
F2 | 5.60 | 224.09 | 4.46 |
C2 | 4.70 | 188.01 | 5.32 |
G2 | 3.36 | 134.56 | 7.43 |
D2 | 4.35 | 174.18 | 5.74 |
H2 | 9.02 | 360.79 | 2.77 |
A3 | 5.81 | 232.50 | 4.30 |
E3 | 2.09 | 83.75 | 11.94 |
B3 | 4.53 | 181.25 | 5.52 |
F3 | 4.22 | 168.76 | 5.93 |
C3 | 4.80 | 191.97 | 5.21 |
G3 | 3.63 | 145.32 | 6.88 |
D3 | 3.61 | 144.24 | 6.93 |
H3 | 2.39 | 95.76 | 10.44 |
A4 | 4.83 | 193.11 | 5.18 |
E4 | 4.57 | 182.86 | 5.47 |
B4 | 9.92 | 396.64 | 2.52 |
F4 | 2.34 | 93.65 | 10.68 |
C4 | 4.01 | 160.57 | 6.23 |
G4 | 4.40 | 176.01 | 5.68 |
D4 | 4.53 | 181.05 | 5.52 |
H4 | 3.63 | 145.19 | 6.89 |
A5 | 5.11 | 204.25 | 4.90 |
E5 | 5.82 | 232.98 | 4.29 |
B5 | 4.21 | 168.60 | 5.93 |
F5 | 6.55 | 261.82 | 3.82 |
C5 | 3.19 | 127.48 | 7.84 |
G5 | 6.35 | 254.06 | 3.94 |
D5 | 3.77 | 150.69 | 6.64 |
H5* | -0.10 | -3.93 | 20* |
A6* | 0.21 | 8.35 | 20* |
E6 | 5.75 | 230.03 | 4.35 |
B6 | 5.73 | 229.25 | 4.36 |
F6 | 3.46 | 138.50 | 7.22 |
C6 | 4.29 | 171.59 | 5.83 |
G6 | 4.10 | 164.04 | 6.10 |
D6 | 2.58 | 103.10 | 9.70 |
H6 | 3.33 | 133.37 | 7.50 |
File:ZhangLab 2 2011-01-11 17hr 27min JHU PAGE size selection.jpg
- perform PAGE analysis to check the size and conc. of DNA library.
File:ZhangLab 2 2011-01-12 10hr 31min PQ SeqLib.jpg
- 0.6 and 0.3ul of DNA was analyzed by PAGE quantification, conc.: 6.55ng/ul or 32.0nM (yield = 6.55ng/ul * 30ul = 196.5ng
- Sequencing : Sample ID: TumorJHU.Ind1to48-Dec18 HL086 run
- After SE sequencing in HL086 run, the amount of each library was normalized based on number of reads and new multiplex libraries were prepared for HiSeq at JHU
- continued on 2011_01_20: [[2]]