Dinh/NOTES/2010-9-1: Difference between revisions
Jump to navigation
Jump to search
>Dinh mNo edit summary |
>Dinh mNo edit summary |
||
(6 intermediate revisions by the same user not shown) | |||
Line 1: | Line 1: | ||
==Prepare DMR#1-7 probe subsets for production PCR:== | |||
{| {{table}} border=1 | {| {{table}} border=1 | ||
| align="center" style="background:#f0f0f0;"|'''DMR-#''' | | align="center" style="background:#f0f0f0;"|'''DMR-#''' | ||
Line 21: | Line 22: | ||
|} | |} | ||
ng/ul -> nM = (ng/ul)*10^6/(146bp*660D/bp) | ng/ul -> nM = (ng/ul)*10^6/(146bp*660D/bp) | ||
==Perform bisulfite conversion on these samples:== | |||
{| {{table}} border=1 | |||
| align="center" style="background:#f0f0f0;"| | |||
| align="center" style="background:#f0f0f0;"|'''Conc(ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Vol to 2 ug''' | |||
| align="center" style="background:#f0f0f0;"|'''Vol''' | |||
| align="center" style="background:#f0f0f0;"|'''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== | |||
'''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 |
Latest revision as of 17:45, 10 September 2010
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