Noi/NOTES/2012-8-23

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Revision as of 23:05, 25 August 2012 by >Noi
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Bisulfite conversion

Lab IDs Conc. (ng/ul) Volume for 2000ng LabID ID H2O Total amount (ng) CT conversion reagent (ul) Total volume (ul)
GL-1 295.8 6.76 GL-1 13.24 2000.00 130.00 150.00
GL-2 220.6 9.07 GL-2 10.93 2000.00 130.00 150.00
GL-3 149.7 13.36 GL-3 6.64 2000.00 130.00 150.00
GL-4 172.3 11.61 GL-4 8.39 2000.00 130.00 150.00
GL-5 285.2 7.01 GL-5 12.99 2000.00 130.00 150.00
GL-6 296.6 6.74 GL-6 13.26 2000.00 130.00 150.00
GL-7 320.0 6.25 GL-7 13.75 2000.00 130.00 150.00
GL-8 339.3 5.89 GL-8 14.11 2000.00 130.00 150.00
GL-9 467.5 4.28 GL-9 15.72 2000.00 130.00 150.00
GL-10 431.7 4.63 GL-10 15.37 2000.00 130.00 150.00
GL-11 349.1 5.73 GL-11 14.27 2000.00 130.00 150.00
GL-12 234.5 8.53 GL-12 11.47 2000.00 130.00 150.00
GL-13** 72.2 20.00 GL-13 0.00 1444.80 130.00 150.00
GL-14 137.3 14.56 GL-14 5.44 2000.00 130.00 150.00
GL-15 207.5 9.64 GL-15 10.36 2000.00 130.00 150.00
GL-16 135.5 14.76 GL-16 5.24 2000.00 130.00 150.00
AL-1 226.6 8.83 AL-1 11.17 2000.00 130.00 150.00
AL-2 375.5 5.33 AL-2 14.67 2000.00 130.00 150.00
AL-3 256.8 7.79 AL-3 12.21 2000.00 130.00 150.00
AL-4 142.1 14.08 AL-4 5.92 2000.00 130.00 150.00
AL-5 140.1 14.27 AL-5 5.73 2000.00 130.00 150.00

330k BSPP capture set up

Subset ID Adjusted Multiplier(2012_05_22) Actual amount of probe required (probe:target ratio, 100:1) Multiplier F Probe concentration (ng/ul) Volume (ul) Volume for 23rxn (ul) Subset ID
DMR.s1 0.25 2.24 0.56 26.2 0.02 0.49 DMR.s1
DMR.s2 0.35 2.24 0.78 30.1 0.03 0.60 DMR.s2
DMR.s3 1.15 18.90 21.74 21.15 1.03 23.64 DMR.s3
DMR.s4 1.00 18.90 18.90 22.34 0.85 19.46 DMR.s4
DMR.s5 4.83 18.90 91.29 29 3.15 72.40 DMR.s5
DMR.Exp1 1.00 18.90 18.90 31.88 0.59 13.64 DMR.Exp1
DMR.Exp2 1.00 18.90 18.90 24.11 0.78 18.03 DMR.Exp2
DMR.Exp3 1.00 18.90 18.90 28.9 0.65 15.04 DMR.Exp3
1.05uM Oligo suppressor-set1 0.125 2.88 1.05uM Oligo suppressor-set1
1.05uM Oligo suppressor-set2 0.125 2.88 1.05uM Oligo suppressor-set2
  • This means that the maximum volume of bis-cvt gDNA is 42.15ul in total volume 55ul
Components 1rxn (ul) 23 rxn mix (ul)
10X AmpLigase Buffer 5.50 126.50
DMR330k probe mix (ul) 7.10 163.30
105nM Oligo suppressor 0.25 5.75
Total volume (ul) 12.85 295.55


Sample IDs Conc. (ng/ul) Volume for 250ng (ul) Sample IDs H2O Total amount of
bis-cvt gDNA (ng)
Probe mix (ul) 10X AmpLigase Buffer 1.05uM Oligo suppressor
set1 &2 (ul)
Total volume (ul)
GL-1 6.30 39.68 GL-1 2.47 250.00 7.10 5.50 0.25 55.00
GL-2 5.00 42.15 GL-2 0.00 210.75 7.10 5.50 0.25 55.00
GL-3 8.92 28.03 GL-3 14.12 250.00 7.10 5.50 0.25 55.00
GL-4 7.14 35.01 GL-4 7.14 250.00 7.10 5.50 0.25 55.00
GL-5* 5.34 42.15 GL-5* 0.00 225.08 7.10 5.50 0.25 55.00
GL-6## 3.97 42.15 GL-6## 0.00 167.34 7.10 5.50 0.25 55.00
GL-7* 5.30 42.15 GL-7* 0.00 223.40 7.10 5.50 0.25 55.00
GL-8* 5.08 42.15 GL-8* 0.00 214.12 7.10 5.50 0.25 55.00
GL-9 5.51 42.15 GL-9 0.00 232.25 7.10 5.50 0.25 55.00
GL-10 5.31 42.15 GL-10 0.00 223.82 7.10 5.50 0.25 55.00
GL-11 4.99 42.15 GL-11 0.00 210.33 7.10 5.50 0.25 55.00
GL-12 4.97 42.15 GL-12 0.00 209.49 7.10 5.50 0.25 55.00
GL-13 7.11 35.16 GL-13 6.99 250.00 7.10 5.50 0.25 55.00
GL-14 7.27 34.39 GL-14 7.76 250.00 7.10 5.50 0.25 55.00
GL-15 8.01 31.21 GL-15 10.94 250.00 7.10 5.50 0.25 55.00
GL-16 7.25 34.48 GL-16 7.67 250.00 7.10 5.50 0.25 55.00
AL-1 5.06 42.15 AL-1 0.00 213.28 7.10 5.50 0.25 55.00
AL-2 5.00 42.15 AL-2 0.00 210.75 7.10 5.50 0.25 55.00
AL-3 5.22 42.15 AL-3 0.00 220.02 7.10 5.50 0.25 55.00
AL-4 7.16 34.92 AL-4 7.23 250.00 7.10 5.50 0.25 55.00
AL-5 8.40 29.76 AL-5 12.39 250.00 7.10 5.50 0.25 55.00
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h 
-> add 3ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP) (08.24.2012, ~5pm)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.

SLN mix solution

Components Stock conc. Unit Final conc. Unit Prepare volume72ul
AmpliTaq Stoffel 10 U/ul 2 U/ul 14.40
AmpLigase 5 U/ul 0.5 U/ul 7.20
dNTP 1000 uM 100 uM 7.20
10x AmpLigase Buffer 10 x 1 x 7.20
H2O 36.00
Total 72.00
  • Add 3ul of SLN mix to each