Noi/NOTES/2014-3-2: Difference between revisions
Jump to navigation
Jump to search
>Noi (Created page with "= DNA methylation assay - MONOD Project (BSPP capture) = http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes#2014 Link to calendar<br> '''Sample list''' : ...") |
>Noi mNo edit summary |
||
Line 8: | Line 8: | ||
* I did bisulfite converssion by usingEZ DNA Methylation-Lightning™ Kit -Zymo and followed the protocol of the kit | * I did bisulfite converssion by usingEZ DNA Methylation-Lightning™ Kit -Zymo and followed the protocol of the kit | ||
=== Bisulfite conversion set up === | === Bisulfite conversion set up === | ||
{| {{table}} class = wikitable | |||
| align="center" style="background:#f0f0f0;"|'''Samples''' | |||
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume for 600ng (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Go to tube''' | |||
| align="center" style="background:#f0f0f0;"|'''H2O''' | |||
| align="center" style="background:#f0f0f0;"|'''Lightning Conv Reagent''' | |||
|- | |||
| CANC_1||47.70||12.58||MN#1||7.42||130.00 | |||
|- | |||
| CANC_2||45.40||13.22||MN#2||6.78||130.00 | |||
|- | |||
| CANC_3||41.40||14.49||MN#3||5.51||130.00 | |||
|- | |||
| CANC_4||47.10||12.74||MN#4||7.26||130.00 | |||
|- | |||
| CANC_5||68.20||8.80||MN#5||11.20||130.00 | |||
|- | |||
| SR-3||62.20||9.65||MN#6||10.35||130.00 | |||
|- | |||
| MA-16||85.00||7.06||MN#7||12.94||130.00 | |||
|- | |||
| MA-17||49.40||12.15||MN#8||7.85||130.00 | |||
|- | |||
| F10||106.00||5.66||MN#9||14.34||130.00 | |||
|- | |||
| G10||108.00||5.56||MN#10||14.44||130.00 | |||
|- | |||
| G11||82.60||7.26||MN#11||12.74||130.00 | |||
|- | |||
| NTC||0||0||MN#12||20.00||130.00 | |||
|} | |||
* 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 | |||
{| {{table}} class = wikitable | |||
| align="center" style="background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="background:#f0f0f0;"|'''Conc. in the Qubit''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''uL used''' | |||
| align="center" style="background:#f0f0f0;"|'''Dilution''' | |||
| align="center" style="background:#f0f0f0;"|'''Sample conc.''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''Yields in 13ul (ng)''' | |||
| align="center" style="background:#f0f0f0;"|'''% recovery''' | |||
|- | |||
| MN_1||175||ng/mL||1||200||34.9||ng/ul||453.7||75.62 | |||
|- | |||
| MN_2||122||ng/mL||1||200||24.5||ug/mL||318.5||53.08 | |||
|- | |||
| MN_3||122||ng/mL||1||200||24.5||ug/mL||318.5||53.08 | |||
|- | |||
| MN_4||87.6||ng/mL||1||200||17.5||ug/mL||227.5||37.92 | |||
|- | |||
| MN_5||108||ng/mL||1||200||21.7||ug/mL||282.1||47.02 | |||
|- | |||
| MN_6||131||ng/mL||1||200||26.2||ug/mL||340.6||56.77 | |||
|- | |||
| MN_7||107||ng/mL||1||200||21.4||ug/mL||278.2||46.37 | |||
|- | |||
| MN_8||71.4||ng/mL||1||200||14.3||ug/mL||185.9||30.98 | |||
|- | |||
| MN_9||61.5||ng/mL||1||200||12.3||ug/mL||159.9||26.65 | |||
|- | |||
| MN_10||173||ng/mL||1||200||34.6||ug/mL||449.8||74.97 | |||
|- | |||
| MN_11||72.9||ng/mL||1||200||14.6||ug/mL||189.8||31.63 | |||
|} | |||
'''Program''' | |||
1. 98°C for 8 minutes | |||
2. 54°C for 60 minutes | |||
3. 4°C hold | |||
== BSPP capture set up == | |||
=== Probe calculation === | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Probe:taeget''' | |||
| align="center" style="background:#f0f0f0;"|'''200:1''' | |||
| align="center" style="background:#f0f0f0;"|'''Unit''' | |||
| align="center" style="background:#f0f0f0;"|'''Calculation''' | |||
|- | |||
| Probe size (number of individual probe in oligo mix)||83447||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 (200:1)||2.195E-17||mole||200 * 1.098E-19mole | |||
|- | |||
| Probe mix MW (size 83447probes, 110nt)||2.788E+09||g/mole||83447 probes * 110bp *303.7Da/bp | |||
|- | |||
| Mass of probe required||6.119E-08||g||2.195E-17mole * 2.788E+09g/mole | |||
|- | |||
| Mass of probe required||61.20||ng|| | |||
|- | |||
| Probe conc||11.9||ng/ul|| | |||
|- | |||
| Volume of probe||5.14||ul|| | |||
|} | |||
==== Probe & AmpLigase Buffer mix ==== | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Components''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''12.5x''' | |||
|- | |||
| 10X AmpLigase Buffer||2.00||25.00 | |||
|- | |||
| MONOD (11.90ng/ul)||5.14||64.25 | |||
|- | |||
| Toal ||7.14|| | |||
|} | |||
<br> | |||
{| {{table}} class = wikitable | |||
| align="center" style="background:#f0f0f0;"|'''Bis-cvt samples''' | |||
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume for 200ng (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Bis-cvt samples''' | |||
| align="center" style="background:#f0f0f0;"|'''H2O (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''MONOD probe (61.20ng)''' | |||
| align="center" style="background:#f0f0f0;"|'''10X AmpLigase Buffer''' | |||
| align="center" style="background:#f0f0f0;"|'''Total (ul)''' | |||
| align="center" style="background:#f0f0f0;"|'''Converted DNA amount (ng)''' | |||
|- | |||
| MN_1||34.90||5.73||MN_1||7.13||5.14||2.00||20.00||200.00 | |||
|- | |||
| MN_2||24.50||8.16||MN_2||4.70||5.14||2.00||20.00||200.00 | |||
|- | |||
| MN_3||24.50||8.16||MN_3||4.70||5.14||2.00||20.00||200.00 | |||
|- | |||
| MN_4||17.50||11.43||MN_4||1.43||5.14||2.00||20.00||200.00 | |||
|- | |||
| MN_5||21.70||9.22||MN_5||3.64||5.14||2.00||20.00||200.00 | |||
|- | |||
| MN_6||26.20||7.63||MN_6||5.23||5.14||2.00||20.00||200.00 | |||
|- | |||
| MN_7||21.40||9.35||MN_7||3.51||5.14||2.00||20.00||200.00 | |||
|- | |||
| MN_8||14.30||13.00||MN_8||0.00||5.14||2.00||20.14||185.90 | |||
|- | |||
| MN_9||12.30||13.00||MN_9||0.00||5.14||2.00||20.14||159.90 | |||
|- | |||
| MN_10||34.60||5.78||MN_10||7.08||5.14||2.00||20.00||200.00 | |||
|- | |||
| MN_11||14.60||13.00||MN_11||0.00||5.14||2.00||20.14||189.80 | |||
|- | |||
| MN_12||0.00||0.00||MN_12||12.86||5.14||2.00||20.00||0.00 | |||
|} | |||
'''Program''' | |||
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h | |||
-> add 3ul SLN mix (2U/ul Hemo Klentaq (NEB); 0.5U/ul AmpLigase; 100uM dNTP) | |||
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold. | |||
* Note: Concentration of Hemo Klentaq was not provided, so I assumeed it has the same concentration as Stoffel Fragment |
Revision as of 16:18, 3 March 2014
DNA methylation assay - MONOD Project (BSPP capture)
[Link to calendar]
Sample list
- - Cancer cell lines (5 samples)
- - Differentiated H1 & H9 (BMP4 treatment, I also included H1 control, 3 samples)
- - Blood samples (3 samples)
Bisulfite conversion
- I did bisulfite converssion by usingEZ DNA Methylation-Lightning™ Kit -Zymo 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 |
CANC_1 | 47.70 | 12.58 | MN#1 | 7.42 | 130.00 |
CANC_2 | 45.40 | 13.22 | MN#2 | 6.78 | 130.00 |
CANC_3 | 41.40 | 14.49 | MN#3 | 5.51 | 130.00 |
CANC_4 | 47.10 | 12.74 | MN#4 | 7.26 | 130.00 |
CANC_5 | 68.20 | 8.80 | MN#5 | 11.20 | 130.00 |
SR-3 | 62.20 | 9.65 | MN#6 | 10.35 | 130.00 |
MA-16 | 85.00 | 7.06 | MN#7 | 12.94 | 130.00 |
MA-17 | 49.40 | 12.15 | MN#8 | 7.85 | 130.00 |
F10 | 106.00 | 5.66 | MN#9 | 14.34 | 130.00 |
G10 | 108.00 | 5.56 | MN#10 | 14.44 | 130.00 |
G11 | 82.60 | 7.26 | MN#11 | 12.74 | 130.00 |
NTC | 0 | 0 | MN#12 | 20.00 | 130.00 |
- 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
Sample | Conc. in the Qubit | ' | uL used | Dilution | Sample conc. | ' | Yields in 13ul (ng) | % recovery |
MN_1 | 175 | ng/mL | 1 | 200 | 34.9 | ng/ul | 453.7 | 75.62 |
MN_2 | 122 | ng/mL | 1 | 200 | 24.5 | ug/mL | 318.5 | 53.08 |
MN_3 | 122 | ng/mL | 1 | 200 | 24.5 | ug/mL | 318.5 | 53.08 |
MN_4 | 87.6 | ng/mL | 1 | 200 | 17.5 | ug/mL | 227.5 | 37.92 |
MN_5 | 108 | ng/mL | 1 | 200 | 21.7 | ug/mL | 282.1 | 47.02 |
MN_6 | 131 | ng/mL | 1 | 200 | 26.2 | ug/mL | 340.6 | 56.77 |
MN_7 | 107 | ng/mL | 1 | 200 | 21.4 | ug/mL | 278.2 | 46.37 |
MN_8 | 71.4 | ng/mL | 1 | 200 | 14.3 | ug/mL | 185.9 | 30.98 |
MN_9 | 61.5 | ng/mL | 1 | 200 | 12.3 | ug/mL | 159.9 | 26.65 |
MN_10 | 173 | ng/mL | 1 | 200 | 34.6 | ug/mL | 449.8 | 74.97 |
MN_11 | 72.9 | ng/mL | 1 | 200 | 14.6 | ug/mL | 189.8 | 31.63 |
Program 1. 98°C for 8 minutes 2. 54°C for 60 minutes 3. 4°C hold
BSPP capture set up
Probe calculation
Probe:taeget | 200:1 | Unit | Calculation |
Probe size (number of individual probe in oligo mix) | 83447 | 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 (200:1) | 2.195E-17 | mole | 200 * 1.098E-19mole |
Probe mix MW (size 83447probes, 110nt) | 2.788E+09 | g/mole | 83447 probes * 110bp *303.7Da/bp |
Mass of probe required | 6.119E-08 | g | 2.195E-17mole * 2.788E+09g/mole |
Mass of probe required | 61.20 | ng | |
Probe conc | 11.9 | ng/ul | |
Volume of probe | 5.14 | ul |
Probe & AmpLigase Buffer mix
Components | Volume (ul) | 12.5x |
10X AmpLigase Buffer | 2.00 | 25.00 |
MONOD (11.90ng/ul) | 5.14 | 64.25 |
Toal | 7.14 |
Bis-cvt samples | Conc. (ng/ul) | Volume for 200ng (ul) | Bis-cvt samples | H2O (ul) | MONOD probe (61.20ng) | 10X AmpLigase Buffer | Total (ul) | Converted DNA amount (ng) |
MN_1 | 34.90 | 5.73 | MN_1 | 7.13 | 5.14 | 2.00 | 20.00 | 200.00 |
MN_2 | 24.50 | 8.16 | MN_2 | 4.70 | 5.14 | 2.00 | 20.00 | 200.00 |
MN_3 | 24.50 | 8.16 | MN_3 | 4.70 | 5.14 | 2.00 | 20.00 | 200.00 |
MN_4 | 17.50 | 11.43 | MN_4 | 1.43 | 5.14 | 2.00 | 20.00 | 200.00 |
MN_5 | 21.70 | 9.22 | MN_5 | 3.64 | 5.14 | 2.00 | 20.00 | 200.00 |
MN_6 | 26.20 | 7.63 | MN_6 | 5.23 | 5.14 | 2.00 | 20.00 | 200.00 |
MN_7 | 21.40 | 9.35 | MN_7 | 3.51 | 5.14 | 2.00 | 20.00 | 200.00 |
MN_8 | 14.30 | 13.00 | MN_8 | 0.00 | 5.14 | 2.00 | 20.14 | 185.90 |
MN_9 | 12.30 | 13.00 | MN_9 | 0.00 | 5.14 | 2.00 | 20.14 | 159.90 |
MN_10 | 34.60 | 5.78 | MN_10 | 7.08 | 5.14 | 2.00 | 20.00 | 200.00 |
MN_11 | 14.60 | 13.00 | MN_11 | 0.00 | 5.14 | 2.00 | 20.14 | 189.80 |
MN_12 | 0.00 | 0.00 | MN_12 | 12.86 | 5.14 | 2.00 | 20.00 | 0.00 |
Program -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 3ul SLN mix (2U/ul Hemo Klentaq (NEB); 0.5U/ul AmpLigase; 100uM dNTP) -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
- Note: Concentration of Hemo Klentaq was not provided, so I assumeed it has the same concentration as Stoffel Fragment