Alice:Exome/2012-12-14

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Revision as of 23:10, 22 January 2013 by >Zsakura2 (Created page with "= WGBS of Xu mouse SCNT and iPSC samples = == End repair == * Total Volume 70ul, sheared DNA 50ul, reaction mix 20ul {| {{table}} border = 1 | align="center" style="background...")
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WGBS of Xu mouse SCNT and iPSC samples

End repair

  • Total Volume 70ul, sheared DNA 50ul, reaction mix 20ul
NEB End repair mix 1x rxn 13x rxn mix
10X End repai buffer 7.00 91.00
NEB End repair enzyme 5.00 65.00
H2O 8.00 104.00
Total 20.00 260.00
- Aliquot to column #1: 
- A1-D1 ~ 42ul
- E1-H1 ~21ul

A-tailing

  • Total volume 50ul
A-tailing reaction mix 1x rxn 13x rxn mix
NEB 10X A-tailing buffer 3.00 39.00
A-tailing enzyme 3.00 39.00
H2O 24.00 312.00
Total 30.00 390.00
- Aliquot to column #2:
- A2-D2 ~ 63ul
- E2-H2 ~31ul

Adapter ligation

  • Total volume 50ul, A-tail reaction mix 40ul, methylation adapters 10ul
Ligation reaction mix 1x rxn 13x rxn mix
5x Quick Ligation Reaction buffer 10.00 130.00
Quick T4 DNA ligase 4.00 52.00
H2O 26.00 338.00
Total 40.00 520.00
- Aliquot to column #3:
- A3-D3 ~ 83 ul
- E3-H3 ~41ul 

Methylation adapter preparation

- Use 10ul of methylated adapter to 5ug gDNA insert
- Use 3ul for 1.5ug DNA insert
- Note: We don't have enough adapter, so I reduced to 2ul
Prepare adapter 2.0ulx 13rxn 26.00
H2O 7.0ulx13rxn 91.00
Total 117.00
- Aliquot to column #4:
- A4-D4 ~ 22 ul
- E4-H4 ~11ul
sample ID N2 Index used)
2A4F1 N2 index 1
2A4F33 N2 index 2
miPS D1 N2 index 3 (not sequenced)
miPS B3 N2 index 4
1E12P20 N2 index 5
nt ES P7 C N2 index 6
nt ES P8 B N2 index 7
NB 1 N2 index 8 (not sequenced)
NB 3 N2 index 9
SCNT B12 N2 index 10
B6 ES N2 index 11
129 ES N2 index 12