Noi/NOTES/2014-6-1

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MONOD V1N3 BSPP capture (repeat sensitivity test)[edit]

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[edit]

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[edit]

  • I did many bisulfite conversion reactions to compare the recovery rate with different inputs
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[edit]

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) 0 0
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
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h 
-> add 1ul SLN mix (2U/ul Hemo Klentaql fragment; 0.5U/ul AmpLigase; 100uM dNTP)
-> 55C 4:40h-> 94C 2min -> add 1.5ul Exo I/III mix (1ul of 20U/ul ExoI & 0.5ul of 200U/ul of ExoIII)-> 37C 2h -> 94C 2min -> 4C hold.

Amplification[edit]

  • I used 10ul of captured DNA in total 75ul PCR reaction.
Components 1x rxn 20 rxn mix
Captured template 10.00 0.00
10uM AmpF6.4Sol 1.50 30.00
10uM AmpR6.3.IndX (X=57-72) 1.50 0.00
2X KAPA SYBR MM 37.50 750.00
H2O 24.50 490.00
Total 75.00
- Aliquot 63.5ul + 1.5ul AmpR6.3 IndX + 10ul template
- Set cycle number maximum at 27X
Program (Eppendorf Realplex)

98C 30s -> (98C 10s -> 58C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x19 -> 72C 3min
Sample Index Sample Index
500:1_5ng_1 Ind_57 1000:1_5ng_1 Ind_65
500:1_5ng_2 Ind_58 1000:1_5ng_2 Ind_66
500:1_10ng_1 Ind_59 1000:1_10ng_1 Ind_67
500:1_10ng_2 Ind_60 1000:1_10ng_2 Ind_68
500:1_25ng_1 Ind_61 1000:1_25ng_1 Ind_69
500:1_25ng_2 Ind_62 1000:1_25ng_2 Ind_70
500:1_50ng_1 Ind_63 1000:1_50ng_1 Ind_71
500:1_50ng_2 Ind_64 1000:1_50ng_2 Ind_72

PAGE verification[edit]

  • 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

  • Dr. Zhang suggested to do sequencing on 5ng and 10ng input.
  • I pooled PCR product by using the gel image intensity to get equimolar ratio of each libraries, including 5ng-1000:1 rep 1 &2 and 10ng-1000:1 rep1 and 2.
  • Purified pooled PCR products with 0.8X AMPure beads and eluted with 40ul EB buffer.
  • Loaded in 2-wells of 5-well 6% TBE gel
File:ZhangLab 2 2014-06-06 09hr 53min PAGE-SS V1N3-5-10ng.jpg
  • Resuspend sequencing libraries in 20ul H2O

Sequencing library ID: NP-BSPP-MNV1N3-5-10ng, Jun1, 2014

- Conc. by Qubit dsDNA HS = 0.644ng/ul (Alan did not have enough room for more libraries in qPCR)
- Run: HL188 (required ~300k/library)
- Length: 375bp
- Index: 65-68 (5ng-rep1, 5ng-rep2, 10ng-rep1, 10ng-rep2)
- They all used probe:target = 1000:1
Index Sample
Ind_65 JK_5ng_1
Ind_66 JK_5ng_2
Ind_67 JK_10ng_1
Ind_68 JK_10ng_2
- JK = Jurkat DNA