Dinh/NOTES/2010-9-1

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Prepare DMR#1-7 probe subsets for production PCR:[edit]

DMR-# ng/ul nM to 10nM vol EB
1 16.8 174.346 1080.95 1018.95
2 13.8 143.213 887.92 825.92
3 9.6 99.626 617.68 555.68
4 8.8 91.324 566.21 504.21
5 10.7 111.042 688.46 626.46
6 9.5 98.589 611.25 549.25
7 12 124.533 772.1 710.1

ng/ul -> nM = (ng/ul)*10^6/(146bp*660D/bp)

Perform bisulfite conversion on these samples:[edit]

Conc(ng/ul) Vol to 2 ug Vol Final Conc (ng/ul) Qubit
hNSC 65.36 30.6 35 -> vacuum centrifuge to ~20ul 76.5
hNiPS2F p23 263.5 7.59 7.59 34.88
HFF-XF 249.27 8.02 8.02 *18.18
FiPS3F-1 198.03 10.1 10.1 26.75
hFiPS4F-7 p9 182.26 10.97 10.97 22.65
KiPS4F-8 567.37 3.53 3.53 54.38
K-MMTA 417.7 4.79 4.79 31.75
hKiPS3F-7 p24 37.59 53.21 55 -> ~20ul *7.78
ASTiPS4F-5 45.51 43.95 45 -> ~20ul 101.94
ASTROCYTES 28.53 70.1 -- ---
ASTiPS4F-4 29.49 67.82 -- ---
HUVEC p2 190.29 10.51 10.51 55.63
HUViPS4F-1 p20 39.55 50.57 55 -> ~20ul 104.75
HUViPS4F-3 p 27.92 71.63 75 -> ~20ul 111.25
  • At least 30ng/ul x 4ul required for capture, repeat bisulfite conversion on these two using more input gDNA

Capture[edit]

Program

   95c 30sec -> cool down to 58C at 0.02C/sec -> 58C 20h 
   -> add 2ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 50uM dNTP) 
   -> 58C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 1h -> 94C 2min -> 4C hold.

SLN

Reagent        1x (ul)     33x (ul)
H2O            0.55        18.15
dNTP(1mM)      0.05        1.65
10xAmpLigase   0.10        3.3
Ligase         0.10        3.3
Stoffel        0.20        6.6

RT_PCR

Reagent        1x (ul)     17x (ul)
Template       10          ---
2xPhusionHF    50          850
AmpF6.3NH2     2           34
AmpR6.3NH2     2           34
SYBR 50x       0.4         6.8
H2O            35.6        605.2