Noi/NOTES/2012-8-26: Difference between revisions

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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)