Noi/NOTES/2014-3-2: Difference between revisions

From ZhangLabWiki
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