Noi/NOTES/2010-12-18

From ZhangLabWiki
Jump to navigation Jump to search

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]]