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