Noi/NOTES/2012-8-26: Difference between revisions
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>Noi (Created page with "* http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''' = WGBS library preparation of low input DNA N37 10 tissues samples using KAPA...") |
>Noi mNo edit summary |
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= WGBS library preparation of low input DNA N37 10 tissues samples using KAPA protocol (from Eppendorf) and epMotion = | = WGBS library preparation of low input DNA N37 10 tissues samples using KAPA protocol (from Eppendorf) and epMotion = | ||
* Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-7-31 | * Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2012-7-31 | ||
== Preparation of enzyme mix for End repair, A-tailing, Ligation, and Methylation adapters == | |||
'''Sample list'''<br> | |||
{| {{table}} border = 1 | |||
| align="center" style="background:#f0f0f0;"|'''Sample IDs''' | |||
| align="center" style="background:#f0f0f0;"|'''Sample code on tubes''' | |||
| align="center" style="background:#f0f0f0;"|'''Well IDs''' | |||
|- | |||
| N37-Cerebellum (CRBL)||N37-1||A1 | |||
|- | |||
| N37-Colon||N37-2||B1 | |||
|- | |||
| N37-Frontal lobe (FL)||N37-3||C1 | |||
|- | |||
| N37-Heart||N37-4||D1 | |||
|- | |||
| N37-Small intestine (SI)||N37-5||E1 | |||
|- | |||
| N37-Liver||N37-6||F1 | |||
|- | |||
| N37-Lung||N37-7||G1 | |||
|- | |||
| N37-Skeletal muscle (SM)||N37-8||H1 | |||
|- | |||
| N37-Pancrease||N37-9||A2 | |||
|- | |||
| N37-Stomach||N37-10||B2 | |||
|}<br> | |||
=== End repair === | |||
Sheared gDNA 200ng in 50ul | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Total reaction in''' | |||
| align="center" style="background:#f0f0f0;"|'''70''' | |||
| align="center" style="background:#f0f0f0;"|'''ul''' | |||
|- | |||
| DNA ||50||ul | |||
|- | |||
| End repair reaction mix ||20||ul | |||
|}<br> | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''KAPA End repair mix''' | |||
| align="center" style="background:#f0f0f0;"|'''1x rxn''' | |||
| align="center" style="background:#f0f0f0;"|'''10x rxn mix''' | |||
|- | |||
| 10X End repai buffer||7.00||70.00 | |||
|- | |||
| KAPA End repair enzyme||3.00||30.00 | |||
|- | |||
| H2O||10.00||100.00 | |||
|- | |||
| Total||20.00||200.00 | |||
|} | |||
* Aliquot to column #1: 20ul (1 & 2: 40ul) | |||
<br> | |||
=== A-tailing === | |||
* Total 30ul/reaction | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''A-tailing reaction mix''' | |||
| align="center" style="background:#f0f0f0;"|'''1x rxn''' | |||
| align="center" style="background:#f0f0f0;"|'''10x rxn mix''' | |||
|- | |||
| KAPA 10X A-tailing buffer||3.00||30.00 | |||
|- | |||
| KAPA A-tailing enzyme||2.00||20.00 | |||
|- | |||
| H2O||25.00||250.00 | |||
|- | |||
| Total||30.00||300.00 | |||
|} | |||
* Aliquot to column #2: 30ul (1 & 2: 60ul) | |||
<br> | |||
=== Adapter ligation === | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Total reaction in''' | |||
| align="center" style="background:#f0f0f0;"|'''50''' | |||
| align="center" style="background:#f0f0f0;"|'''ul''' | |||
|- | |||
| Ligation reaction mix||40||ul | |||
|- | |||
| Diluted Methylation adapters||10||ul | |||
|}<br> | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Ligation reaction mix''' | |||
| align="center" style="background:#f0f0f0;"|'''1x rxn''' | |||
| align="center" style="background:#f0f0f0;"|'''10x rxn mix''' | |||
|- | |||
| KAPA 5X Ligation buffer||10.00||100.00 | |||
|- | |||
| KAPA DNA ligase||3.00||30.00 | |||
|- | |||
| H2O||27.00||270.00 | |||
|- | |||
| Total||40.00||400.00 | |||
|} | |||
* Aliquot ligation reaction mix to column #3: 40ul (1 & 2: 80ul) | |||
'''Methylation adapter preparation'''<br> | |||
{| {{table}} | |||
| Prepare adapter||0.2ulx 10rxn||2.00 | |||
|- | |||
| H2O||9.8ulx10rxn||98.00 | |||
|- | |||
| Total||||100 | |||
|} | |||
* Aliquot diluted methylation adapter to column #4: 10ul (1 & 2: 20ul) |
Revision as of 18:51, 26 August 2012
WGBS library preparation of low input DNA N37 10 tissues samples using KAPA protocol (from Eppendorf) and epMotion
Preparation of enzyme mix for End repair, A-tailing, Ligation, and Methylation adapters
Sample list
Sample IDs | Sample code on tubes | Well IDs |
N37-Cerebellum (CRBL) | N37-1 | A1 |
N37-Colon | N37-2 | B1 |
N37-Frontal lobe (FL) | N37-3 | C1 |
N37-Heart | N37-4 | D1 |
N37-Small intestine (SI) | N37-5 | E1 |
N37-Liver | N37-6 | F1 |
N37-Lung | N37-7 | G1 |
N37-Skeletal muscle (SM) | N37-8 | H1 |
N37-Pancrease | N37-9 | A2 |
N37-Stomach | N37-10 | B2 |
End repair
Sheared gDNA 200ng in 50ul
Total reaction in | 70 | ul |
DNA | 50 | ul |
End repair reaction mix | 20 | ul |
KAPA End repair mix | 1x rxn | 10x rxn mix |
10X End repai buffer | 7.00 | 70.00 |
KAPA End repair enzyme | 3.00 | 30.00 |
H2O | 10.00 | 100.00 |
Total | 20.00 | 200.00 |
- Aliquot to column #1: 20ul (1 & 2: 40ul)
A-tailing
- Total 30ul/reaction
A-tailing reaction mix | 1x rxn | 10x rxn mix |
KAPA 10X A-tailing buffer | 3.00 | 30.00 |
KAPA A-tailing enzyme | 2.00 | 20.00 |
H2O | 25.00 | 250.00 |
Total | 30.00 | 300.00 |
- Aliquot to column #2: 30ul (1 & 2: 60ul)
Adapter ligation
Total reaction in | 50 | ul |
Ligation reaction mix | 40 | ul |
Diluted Methylation adapters | 10 | ul |
Ligation reaction mix | 1x rxn | 10x rxn mix |
KAPA 5X Ligation buffer | 10.00 | 100.00 |
KAPA DNA ligase | 3.00 | 30.00 |
H2O | 27.00 | 270.00 |
Total | 40.00 | 400.00 |
- Aliquot ligation reaction mix to column #3: 40ul (1 & 2: 80ul)
Methylation adapter preparation
Prepare adapter | 0.2ulx 10rxn | 2.00 |
H2O | 9.8ulx10rxn | 98.00 |
Total | 100 |
- Aliquot diluted methylation adapter to column #4: 10ul (1 & 2: 20ul)