Noi/NOTES/2014-6-1: Difference between revisions
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>Noi |
>Noi |
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* I will add experimental procedures later | * I will add experimental procedures later | ||
== Bisulfite conversion == | == Bisulfite conversion == | ||
{| {{table}} | {| {{table}} border = 1 | ||
| align="center" style="background:#f0f0f0;"|'''Mix''' | | align="center" style="background:#f0f0f0;"|'''Mix''' | ||
| align="center" style="background:#f0f0f0;"|'''82.1ng/ul JK DNA''' | | align="center" style="background:#f0f0f0;"|'''82.1ng/ul JK DNA''' | ||
Line 21: | Line 21: | ||
| 200ng||10.23||73.77||20.00 | | 200ng||10.23||73.77||20.00 | ||
|} | |} | ||
{| {{table}} | {| {{table}} border = 1 | ||
| align="center" style="background:#f0f0f0;"|'''Sample''' | | align="center" style="background:#f0f0f0;"|'''Sample''' | ||
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | ||
| align="center" style="background:#f0f0f0;"|'''Amount (ng)''' | | align="center" style="background:#f0f0f0;"|'''Amount (ng)''' | ||
| align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | ||
| align="center" style="background:#f0f0f0;"|'''H2O (ul)''' | | align="center" style="width:60px;background:#f0f0f0;"|'''H2O (ul)''' | ||
| align="center" style="background:#f0f0f0;"|'''Lightnig Conversion reagent''' | | align="center" style="width:60px;background:#f0f0f0;"|'''Lightnig Conversion reagent''' | ||
| align="center" style="background:#f0f0f0;"|'''Total''' | | align="center" style="width:60px;background:#f0f0f0;"|'''Total''' | ||
|- | |- | ||
| JK-1||82.1||25||0.30||19.70||130||150.00 | | JK-1||82.1||25||0.30||19.70||130||150.00 |
Revision as of 00:47, 5 June 2014
MONOD V1N3 BSPP capture (repeat sensitivity test)
Link to calendar: [[1]]
- I repeated MONOD V1N3 capture experiment on 3014-05-29: [[2]]
- I reduced capture volume to 10ul.
- I reduced incubation time after adding SLN mix between 4-6h since I think that 20h is not necessary for polymerization and may cause more back ground if DNA template level is too low.
- I will add experimental procedures later
Bisulfite conversion
Mix | 82.1ng/ul JK DNA | H2O | Aliquot |
25ng | 1.28 | 82.72 | 20.00 |
50ng | 2.56 | 81.44 | 20.00 |
100ng | 5.12 | 78.88 | 20.00 |
200ng | 10.23 | 73.77 | 20.00 |
Sample | Conc. (ng/ul) | Amount (ng) | Volume (ul) | H2O (ul) | Lightnig Conversion reagent | Total |
JK-1 | 82.1 | 25 | 0.30 | 19.70 | 130 | 150.00 |
JK-2 | 82.1 | 25 | 0.30 | 19.70 | 130 | 150.00 |
JK-3 | 82.1 | 25 | 0.30 | 19.70 | 130 | 150.00 |
JK-4 | 82.1 | 25 | 0.30 | 19.70 | 130 | 150.00 |
JK-5 | 82.1 | 50 | 0.61 | 19.39 | 130 | 150.00 |
JK-6 | 82.1 | 50 | 0.61 | 19.39 | 130 | 150.00 |
JK-7 | 82.1 | 50 | 0.61 | 19.39 | 130 | 150.00 |
JK-8 | 82.1 | 50 | 0.61 | 19.39 | 130 | 150.00 |
JK-9 | 82.1 | 100 | 1.22 | 18.78 | 130 | 150.00 |
JK-10 | 82.1 | 100 | 1.22 | 18.78 | 130 | 150.00 |
JK-11 | 82.1 | 100 | 1.22 | 18.78 | 130 | 150.00 |
JK-12 | 82.1 | 100 | 1.22 | 18.78 | 130 | 150.00 |
JK-13 | 82.1 | 200 | 2.44 | 17.56 | 130 | 150.00 |
JK-14 | 82.1 | 200 | 2.44 | 17.56 | 130 | 150.00 |
JK-15 | 82.1 | 200 | 2.44 | 17.56 | 130 | 150.00 |
JK-16 | 82.1 | 200 | 2.44 | 17.56 | 130 | 150.00 |
- Elute with 10ul Elution buffer
- Use 1ul for Qubit ssDNA assay
Qubit ssDNA assay results
Sample | Conc. in the Qubit | Unit | uL used | Dilution | Sample conc. (ng/ul) | Yield in 9ul (ng) | Input DNA (ng) | % Recovery |
JK-1 | 3.8 | ng/mL | 1 | 200 | 0.760 | 6.84 | 25 | 27.36 |
JK-2 | 4.9 | ng/mL | 1 | 200 | 0.980 | 8.82 | 25 | 35.28 |
JK-3 | 2.5 | ng/mL | 1 | 200 | 0.500 | 4.50 | 25 | 18.00 |
JK-4 | 5.11 | ng/mL | 1 | 200 | 1.022 | 9.20 | 25 | 36.79 |
JK-5 | 10.8 | ng/mL | 1 | 200 | 2.160 | 19.44 | 50 | 38.88 |
JK-6 | 9.06 | ng/mL | 1 | 200 | 1.812 | 16.31 | 50 | 32.62 |
JK-7 | 12.3 | ng/mL | 1 | 200 | 2.460 | 22.14 | 50 | 44.28 |
JK-8 | 11.2 | ng/mL | 1 | 200 | 2.240 | 20.16 | 50 | 40.32 |
JK-9 | 25 | ng/mL | 1 | 200 | 5.000 | 45.00 | 100 | 45.00 |
JK-10 | 26.7 | ng/mL | 1 | 200 | 5.340 | 48.06 | 100 | 48.06 |
JK-11 | 28.4 | ng/mL | 1 | 200 | 5.680 | 51.12 | 100 | 51.12 |
JK-12 | 24.3 | ng/mL | 1 | 200 | 4.860 | 43.74 | 100 | 43.74 |
JK-13 | 90.1 | ng/mL | 1 | 200 | 18.020 | 162.18 | 200 | 81.09 |
JK-14 | 89 | ng/mL | 1 | 200 | 17.800 | 160.20 | 200 | 80.10 |
JK-15 | 79.7 | ng/mL | 1 | 200 | 15.940 | 143.46 | 200 | 71.73 |
JK-16 | 85.4 | ng/mL | 1 | 200 | 17.080 | 153.72 | 200 | 76.86 |
BSPP capture set up
strip 1 | 5:500 | 5:500 | 10:500 | 10:500 | 25:500 | 25:500 | 50:500 | 50:500 |
BIS-DNA required (ng) | 5 | 5 | 10 | 10 | 25 | 25 | 50 | 50 |
JK # | JK-1 | JK-2 | JK-5 | JK-6 | JK-9 | JK-10 | JK-13 | JK-14 |
Conc. | 0.760 | 0.980 | 2.160 | 1.812 | 5.000 | 5.340 | 18.020 | 17.800 |
Volume (ul) | 6.58 | 5.10 | 4.63 | 5.52 | 5.00 | 4.68 | 2.77 | 2.81 |
H2O (ul) | 0.27 | 1.75 | 2.07 | 1.18 | 1.25 | 1.57 | 2.72 | 2.69 |
V1N3 amount (ng) | 0.11 | 0.11 | 0.22 | 0.22 | 0.55 | 0.55 | 1.10 | 1.10 |
V1N3 conc. (ng/ul) | 0.73 | 0.73 | 0.73 | 0.73 | 0.73 | 0.73 | 0.73 | 0.73 |
V1N3 volume (ul) | 0.15 | 0.15 | 0.30 | 0.30 | 0.75 | 0.75 | 1.50 | 1.50 |
10X Ampligase Buffer | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
H2O in Mix | 2.00 | 2.00 | 2.00 | 2.00 | 2.00 | 2.00 | 2.00 | 2.00 |
Total | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 |
strip 2 | 5:1000 | 5:1000 | 10:1000 | 10:1000 | 25:1000 | 25:1000 | 50:1000 | 50:1000 |
BIS-DNA required (ng) | 5 | 5 | 10 | 10 | 25 | 25 | 50 | 50 |
JK # | JK-4 | JK-5 | JK-7 | JK-8 | JK-11 | JK-12 | JK-15 | JK-16 |
Conc. | 1.022 | 2.160 | 2.460 | 2.240 | 5.680 | 4.860 | 15.940 | 17.080 |
Volume (ul) | 4.89 | 2.31 | 4.07 | 4.46 | 4.40 | 5.14 | 3.14 | 2.93 |
H2O (ul) | 1.81 | 4.38 | 2.33 | 1.93 | 1.09 | 0.35 | 0.85 | 1.06 |
V1N3 amount (ng) | 0.22 | 0.22 | 0.44 | 0.44 | 1.10 | 1.10 | 2.20 | 2.20 |
V1N3 conc. (ng/ul) | 0.73 | 0.73 | 0.73 | 0.73 | 0.73 | 0.73 | 0.73 | 0.73 |
V1N3 volume (ul) | 0.30 | 0.30 | 0.60 | 0.60 | 1.50 | 1.50 | 3.01 | 3.01 |
10X Ampligase Buffer | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 | 1.00 |
H2O in Mix | 2.00 | 2.00 | 2.00 | 2.00 | 2.00 | 2.00 | 2.00 | 2.00 |
Total | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 | 10.00 |
strip 3 | NTC:500 | NTC:1000 |
BIS-DNA required (ng) | 5 | 5 |
JK # | 0 | 0 |
Conc. | 0 | 0 |
Volume (ul) | 0.00 | 0.00 |
H2O (ul) | 5.50 | 3.99 |
V1N3 amount (ng) | 1.10 | 2.20 |
V1N3 conc. (ng/ul) | 0.73 | 0.73 |
V1N3 volume (ul) | 1.50 | 3.01 |
10X Ampligase Buffer | 1.00 | 1.00 |
H2O in Mix | 2.00 | 2.00 |
Total | 10.00 | 10.00 |
V1N3 & Ampligase Buffer mix
Mix 0.11 | 1x | 4x |
Diluted V1N3 | 0.15 | 0.60 |
10X Ampligase Buffer | 1.00 | 4.00 |
H2O | 2.00 | 8.00 |
Sum | 3.15 |
Mix 0.22 | 1x | 6x |
Diluted V1N3 | 0.30 | 1.80 |
10X Ampligase Buffer | 1.00 | 6.00 |
H2O | 2.00 | 12.00 |
Sum | 3.30 |
Mix 0.44 | 1x | 4x |
Diluted V1N3 | 0.60 | 2.40 |
10X Ampligase Buffer | 1.00 | 4.00 |
H2O | 2.00 | 8.00 |
Sum | 3.60 |
Mix 0.55 | 1x | 4x |
Diluted V1N3 | 0.75 | 3.00 |
10X Ampligase Buffer | 1.00 | 4.00 |
H2O | 2.00 | 8.00 |
Sum | 3.75 |
Mix 1.1 | 1x | 6x |
Diluted V1N3 | 1.50 | 9.00 |
10X Ampligase Buffer | 1.00 | 6.00 |
H2O | 2.00 | 12.00 |
Sum | 4.50 |
Mix 2.2 | 1x | 4x |
Diluted V1N3 | 3.01 | 12.04 |
10X Ampligase Buffer | 1.00 | 4.00 |
H2O | 2.00 | 8.00 |
Sum | 6.01 |
PAGE verification
- Loaded 4ul of PCR products in 6% TBE gel
File:ZhangLab 2 2014-06-03 16hr 20min V1N3 500to1.jpg
File:ZhangLab 2 2014-06-03 16hr 20min V1N3 1000to1.jpg