Noi/NOTES/2013-7-19: Difference between revisions

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(Created page with "= DNA methylation assay for Blueprint project = * http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''' {| {{table}} | align="center" ...")
 
>Noi
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= DNA methylation assay for Blueprint project =
= DNA methylation assay for Blueprint project =
* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']]
* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']]
== Bisulfite conversion ==
* I did bisulfite converssion by using EZ-96 DNA Methylation-Lightning™ MagPrep - Zymo and followed the protocol of the kit
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Sample IDs'''
| align="center" style="background:#f0f0f0;"|'''Sample IDs'''
Line 79: Line 81:
| 34||B5 (12878)||59.00||8.47||11.53||131.00||151.00||500.00
| 34||B5 (12878)||59.00||8.47||11.53||131.00||151.00||500.00
|}
|}
''' Program '''<br>
1. 98°C for 8 minutes
2. 54°C for 60 minutes
3. 4°C hold
* Eluted bisulfite converted DNA form the bead with 20ul H2O
* I used 1ul for ssDNA Qubit assay (200x dilution). The volume left after assay ~15ul.
==== ssDNA Qubit assay result ====
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Sample IDs'''
| align="center" style="background:#f0f0f0;"|'''WellIDs'''
| align="center" style="background:#f0f0f0;"|'''Start amount (ng)'''
| align="center" style="background:#f0f0f0;"|'''Sample Concentration (ng/ul)'''
| align="center" style="background:#f0f0f0;"|'''Yield in 17ul (ng)'''
| align="center" style="background:#f0f0f0;"|'''Yield left after Qubit (15ul)'''
| align="center" style="background:#f0f0f0;"|'''% Recovery in 17ul'''
|-
| 1||A1||500.00||21.00||357.00||315.00||71.40
|-
| 2||B1||500.00||24.40||414.80||366.00||82.96
|-
| 3||C1||500.00||19.10||324.70||286.50||64.94
|-
| 4||D1||500.00||22.60||384.20||339.00||76.84
|-
| 5||E1||500.00||20.60||350.20||309.00||70.04
|-
| 6||F1||500.00||21.20||360.40||318.00||72.08
|-
| 7||G1||500.00||22.00||374.00||330.00||74.80
|-
| 8||H1||500.00||22.20||377.40||333.00||75.48
|-
| 9||A2||500.00||20.60||350.20||309.00||70.04
|-
| 10||B2||500.00||24.00||408.00||360.00||81.60
|-
| 11||C2||500.00||19.06||324.02||285.90||64.80
|-
| 12||D2||500.00||18.78||319.26||281.70||63.85
|-
| 13||E2||102.09||2.06||35.02||30.90||34.30
|-
| 14||F2||331.71||8.08||137.36||121.20||41.41
|-
| 15||G2||240.69||6.50||110.50||97.50||45.91
|-
| 16||H2||450.00||12.32||209.44||184.80||46.54
|-
| 17||A3||500.00||11.56||196.52||173.40||39.30
|-
| 18||B3||500.00||12.24||208.08||183.60||41.62
|-
| 19||C3||500.00||11.44||194.48||171.60||38.90
|-
| 20||D3||405.71||7.92||134.64||118.80||33.19
|-
| 21||E3||364.57||8.10||137.70||121.50||37.77
|-
| 22||F3||500.00||11.38||193.46||170.70||38.69
|-
| 23||G3||500.00||13.96||237.32||209.40||47.46
|-
| 24||H3||500.00||19.72||335.24||295.80||67.05
|-
| 25||A4||500.00||21.20||360.40||318.00||72.08
|-
| 26||B4||392.00||23.00||391.00||345.00||99.74
|-
| 27||C4||486.29||23.80||404.60||357.00||83.20
|-
| 28||D4||473.14||31.40||533.80||471.00||112.82
|-
| 29||E4||500.00||20.80||353.60||312.00||70.72
|-
| 30||F4||500.00||10.46||177.82||156.90||35.56
|-
| 31||G4||500.00||47.00||799.00||705.00||159.80
|-
| 32||H4||500.00||56.00||952.00||840.00||190.40
|-
| 33||A5 (12878)||500.00||24.00||408.00||360.00||81.60
|-
| 34||B5 (12878)||500.00||24.60||418.20||369.00||83.64
|}
* There are few sample have %recovery >100%. I repeat measurement again and got the same concentration. This is possible that I used too small volume when assay gDNA concentration, and there is some error of pipetting.
* Most of sample start with the same amount of gDNA (500ng) have roughly close concentration of ssDNA after bisulfite conversion.

Revision as of 17:29, 20 July 2013

DNA methylation assay for Blueprint project

Bisulfite conversion

  • I did bisulfite converssion by using EZ-96 DNA Methylation-Lightning™ MagPrep - Zymo and followed the protocol of the kit
Sample IDs WellIDs Conc. (ng/ul) Volume (ul) H2O (ul) CT conversion reagent (ul) Total volume (ul) Total amount (ng)
1 A1 37.71 13.26 6.74 130.00 150.00 500.00
2 B1 39.43 12.68 7.32 130.00 150.00 500.00
3 C1 50.57 9.89 10.11 130.00 150.00 500.00
4 D1 34.86 14.34 5.66 130.00 150.00 500.00
5 E1 56.00 8.93 11.07 130.00 150.00 500.00
6 F1 44.29 11.29 8.71 130.00 150.00 500.00
7 G1 73.71 6.78 13.22 130.00 150.00 500.00
8 H1 29.71 16.83 3.17 130.00 150.00 500.00
9 A2 40.29 12.41 7.59 130.00 150.00 500.00
10 B2 33.43 14.96 5.04 130.00 150.00 500.00
11 C2 44.86 11.15 8.85 130.00 150.00 500.00
12 D2 52.86 9.46 10.54 130.00 150.00 500.00
**13 E2 11.34 9.00 11.00 130.00 150.00 102.09
*14 F2 36.86 9.00 11.00 130.00 150.00 331.71
**15 G2 26.74 9.00 11.00 130.00 150.00 240.69
*16 H2 50.00 9.00 11.00 130.00 150.00 450.00
17 A3 34.00 14.71 5.29 130.00 150.00 500.00
18 B3 31.14 16.06 3.94 130.00 150.00 500.00
19 C3 26.46 18.90 1.10 130.00 150.00 500.00
*20 D3 20.29 20.00 0.00 130.00 150.00 405.71
*21 E3 18.23 20.00 0.00 130.00 150.00 364.57
22 F3 29.43 16.99 3.01 130.00 150.00 500.00
23 G3 47.71 10.48 9.52 130.00 150.00 500.00
24 H3 32.57 15.35 4.65 130.00 150.00 500.00
25 A4 29.14 17.16 2.84 130.00 150.00 500.00
*26 B4 19.60 20.00 0.00 130.00 150.00 392.00
27 C4 24.31 20.00 0.00 130.00 150.00 486.29
28 D4 23.66 20.00 0.00 130.00 150.00 473.14
29 E4 47.43 10.54 9.46 130.00 150.00 500.00
30 F4 76.86 6.51 13.49 130.00 150.00 500.00
31 G4 40.00 12.50 7.50 130.00 150.00 500.00
32 H4 32.29 15.49 4.51 130.00 150.00 500.00
33 A5 (12878) 58.00 8.62 11.38 130.00 150.00 500.00
34 B5 (12878) 59.00 8.47 11.53 131.00 151.00 500.00
 Program 
1. 98°C for 8 minutes 2. 54°C for 60 minutes 3. 4°C hold
  • Eluted bisulfite converted DNA form the bead with 20ul H2O
  • I used 1ul for ssDNA Qubit assay (200x dilution). The volume left after assay ~15ul.

ssDNA Qubit assay result

Sample IDs WellIDs Start amount (ng) Sample Concentration (ng/ul) Yield in 17ul (ng) Yield left after Qubit (15ul) % Recovery in 17ul
1 A1 500.00 21.00 357.00 315.00 71.40
2 B1 500.00 24.40 414.80 366.00 82.96
3 C1 500.00 19.10 324.70 286.50 64.94
4 D1 500.00 22.60 384.20 339.00 76.84
5 E1 500.00 20.60 350.20 309.00 70.04
6 F1 500.00 21.20 360.40 318.00 72.08
7 G1 500.00 22.00 374.00 330.00 74.80
8 H1 500.00 22.20 377.40 333.00 75.48
9 A2 500.00 20.60 350.20 309.00 70.04
10 B2 500.00 24.00 408.00 360.00 81.60
11 C2 500.00 19.06 324.02 285.90 64.80
12 D2 500.00 18.78 319.26 281.70 63.85
13 E2 102.09 2.06 35.02 30.90 34.30
14 F2 331.71 8.08 137.36 121.20 41.41
15 G2 240.69 6.50 110.50 97.50 45.91
16 H2 450.00 12.32 209.44 184.80 46.54
17 A3 500.00 11.56 196.52 173.40 39.30
18 B3 500.00 12.24 208.08 183.60 41.62
19 C3 500.00 11.44 194.48 171.60 38.90
20 D3 405.71 7.92 134.64 118.80 33.19
21 E3 364.57 8.10 137.70 121.50 37.77
22 F3 500.00 11.38 193.46 170.70 38.69
23 G3 500.00 13.96 237.32 209.40 47.46
24 H3 500.00 19.72 335.24 295.80 67.05
25 A4 500.00 21.20 360.40 318.00 72.08
26 B4 392.00 23.00 391.00 345.00 99.74
27 C4 486.29 23.80 404.60 357.00 83.20
28 D4 473.14 31.40 533.80 471.00 112.82
29 E4 500.00 20.80 353.60 312.00 70.72
30 F4 500.00 10.46 177.82 156.90 35.56
31 G4 500.00 47.00 799.00 705.00 159.80
32 H4 500.00 56.00 952.00 840.00 190.40
33 A5 (12878) 500.00 24.00 408.00 360.00 81.60
34 B5 (12878) 500.00 24.60 418.20 369.00 83.64
  • There are few sample have %recovery >100%. I repeat measurement again and got the same concentration. This is possible that I used too small volume when assay gDNA concentration, and there is some error of pipetting.
  • Most of sample start with the same amount of gDNA (500ng) have roughly close concentration of ssDNA after bisulfite conversion.