Noi/NOTES/2014-3-30
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DNA methylation assay - MONOD Project, MONOD_V3_12k probe set (BSPP capture)
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Sample list
- Sample list will be the same as experiment in 2013-03-02
- - Cancer cell lines (5 samples)
- - Differentiated H1 & H9 (BMP4 treatment, I also included H1 control, 3 samples)
- - Blood samples (3 samples)
- I wanted to compare Hemo KlenTaq and Stoffel Fragment, so I will include one of cancer sample with Stoffel Fragment.
Sample IDs tracking
- Refer to previous experiment on 2014-03-02
- Probe preparation
Bisulfite conversion
- * I did bisulfite converssion by usingEZ DNA Methylation-Lightning™ Kit -Zymo (lot Mar 2014) and followed the protocol of the kit
Bisulfite conversion set up
Samples | Conc. (ng/ul) | Volume for 600ng (ul) | Go to tube | H2O | Lightning Conv Reagent | Total (ul) | Samples |
CANC_1 | 47.70 | 12.58 | MN_1 | 7.42 | 130.00 | 150.00 | CANC_1 |
CANC_2 | 45.40 | 13.22 | MN_2 | 6.78 | 130.00 | 150.00 | CANC_2 |
CANC_3 | 41.40 | 14.49 | MN_3 | 5.51 | 130.00 | 150.00 | CANC_3 |
CANC_4 | 47.10 | 12.74 | MN_4 | 7.26 | 130.00 | 150.00 | CANC_4 |
CANC_5 | 68.20 | 8.80 | MN_5 | 11.20 | 130.00 | 150.00 | CANC_5 |
SR-3 | 62.20 | 9.65 | MN_6 | 10.35 | 130.00 | 150.00 | SR-3 |
MA-16 | 85.00 | 7.06 | MN_7 | 12.94 | 130.00 | 150.00 | MA-16 |
MA-17 | 49.40 | 12.15 | MN_8 | 7.85 | 130.00 | 150.00 | MA-17 |
F10 | 106.00 | 5.66 | MN_9 | 14.34 | 130.00 | 150.00 | F10 |
G10 | 108.00 | 5.56 | MN_10 | 14.44 | 130.00 | 150.00 | G10 |
G11 | 82.60 | 7.26 | MN_11 | 12.74 | 130.00 | 150.00 | G11 |
NTC_HemoKlenTaq | 0 | 0 | MN_12 | 20.00 | 130.00 | 150.00 | NTC |
CANC_1 | 47.70 | 12.58 | MN_13 | 7.42 | 130.00 | 150.00 | CANC_1 |
NTC_Stoffel | 0 | 0 | MN_14 | 20.00 | 130.00 | 150.00 | NTC |
Program 1. 98°C for 8 minutes 2. 54°C for 60 minutes 3. 4°C hold
- I elute with Elution buffer 16ul
- I used 1ul for ssDNA Qubit assay. The volume left after assay ~13ul.
Qubit ss DNA assay
- Dilute 200X (add 1ul of bis-cvt DNA in total 200 assay volume
BSPP capture set up
Probe calculation
MONOD_V3_12k, size 12,000
Probe:taeget 500:1 Unit Calculation Probe size (number of individual probe in oligo mix) 12000 probes Human bis-cvt gDNA required 200 ng Human gDNA MW 1.822E+12 g/mole 3.0E+09 * 607.4Da/bp Human gDNA( 200ng) 1.098E-19 mole 200E-09g / 1.822E+12g/mole Probe required (500:1) 5.488E-17 mole 500 * 1.098E-19mole Probe mix MW (size12,000probes, 110nt) 4.018E+08 g/mole 12,000 probes * (110bp *303.7Da/bp+79Da) Mass of probe required 2.205E-08 g 5.488E-17mole * 4.018E+08g/mole Mass of probe required 22.05 ng Probe conc ng/ul Volume of probe ul