Noi/NOTES/2014-3-30

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DNA methylation assay - MONOD Project, MONOD_V3_12k probe set (BSPP capture)

[Link to calendar]
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

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

Probe & AmpLigase Buffer mix