Noi/NOTES/2014-6-1

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Revision as of 23:33, 4 June 2014 by >Noi
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MONOD V1N3 BSPP capture (repeat sensitivity test)

Link to calendar: [[1]]

  • I repeated MONOD V1N3 capture experiment on 3014-05-29.
  • 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