Noi/NOTES/2013-7-19: Difference between revisions
Jump to navigation
Jump to search
>Noi (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 mNo edit summary |
||
Line 1: | Line 1: | ||
= 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.