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