Noi/NOTES/2012-8-1: Difference between revisions
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* Loaded 2.5 ul of bis-cvt DNA and 2.5ul of amplified amplicons | * Loaded 2.5 ul of bis-cvt DNA and 2.5ul of amplified amplicons | ||
[[File:ZhangLab_2 2012-08-02 11hr 31min-HAPMAP-bis-test.jpg| 400px]] <br> | [[File:ZhangLab_2 2012-08-02 11hr 31min-HAPMAP-bis-test.jpg| 400px]] <br> | ||
{| {{table}} border = 1 | |||
| width="160pt" align="center" style="background:#f0f0f0;"|'''Components''' | |||
| width="60pt" align="center" style="background:#f0f0f0;"|'''1 rxn''' | |||
| align="center" style="background:#f0f0f0;"|'''26rxn mix''' | |||
|- | |||
| DNA template||10.00||0.00 | |||
|- | |||
| 10uM PCR_F||2.00||52.00 | |||
|- | |||
| 10uM PCR_R.Indx (1-8)||2.00||0.00 | |||
|- | |||
| 2x iQ Supermix||50.00||1,300.00 | |||
|- | |||
| H2O||36.00||936.00 | |||
|} | |||
* Aliquot 88ul, add 2ul of 10uM PCR_R Ind (Ind1-8), 10ul of bis-cvt DNA template | |||
'''Program'''<br> | |||
98C 2min (98C 15sec -> 64C 20sec -> 72C 30sec) x10cycles -> 72C 3min -> 15CHold | |||
{| {{table}} | {| {{table}} | ||
| width="80pt" align="center" style="background:#f0f0f0;"|'''Sample ID''' | | width="80pt" align="center" style="background:#f0f0f0;"|'''Sample ID''' |
Revision as of 01:36, 3 August 2012
WGBS sequencing library preparation using KAPA protocol (Eppendorf) and epMotion (Repeat!)
- The step of library sequencing prep will be filled soon.
Amplification of bisulfite sequencing llibraries with N2 barcoded primers
- Test if the adapter ligation reaction worked before amplify the libraries with barcoded primers
- Loaded 2.5 ul of bis-cvt DNA and 2.5ul of amplified amplicons
File:ZhangLab 2 2012-08-02 11hr 31min-HAPMAP-bis-test.jpg
Components | 1 rxn | 26rxn mix |
DNA template | 10.00 | 0.00 |
10uM PCR_F | 2.00 | 52.00 |
10uM PCR_R.Indx (1-8) | 2.00 | 0.00 |
2x iQ Supermix | 50.00 | 1,300.00 |
H2O | 36.00 | 936.00 |
- Aliquot 88ul, add 2ul of 10uM PCR_R Ind (Ind1-8), 10ul of bis-cvt DNA template
Program
98C 2min (98C 15sec -> 64C 20sec -> 72C 30sec) x10cycles -> 72C 3min -> 15CHold
Sample ID | Labeling | Indx |
WGBS.GM12891 | #1 | PCR_R.N2Indx1 |
WGBS.GM12892 | #2 | PCR_R.N2Indx2 |
WGBS.GM10861 | #3 | PCR_R.N2Indx3 |
WGBS.GM11994 | #4 | PCR_R.N2Indx4 |
WGBS.GM11995 | #5 | PCR_R.N2Indx5 |
WGBS.GM18506 | #6 | PCR_R.N2Indx6 |
WGBS.GM18507 | #7 | PCR_R.N2Indx7 |
WGBS.GM18505 | #8 | PCR_R.N2Indx8 |
- Purified sequencing libraries with 0.8x vol AMPure bead with epMotion (3 strips), eluted with 25ul EB for each well -> total volume at the end ~70ul
File:ZhangLab 2 2012-08-02 16hr 05min bead-HAPMAP-WGBS.jpg
PAGE quantification of 400-600bp fragments
- Perform PAGE size-selection of the size range 400-600bp for a small scale sequencing. Then pooled 60ng of each library and performed PAGE size-selection.
File:ZhangLab 2 2012-08-02 16hr 05min bead-HAPMAP-WGBS-400-600bp PQ.jpg
Lab IDs | Final conc.
of 400-600bp (ng/ul) |
Total yields in 70ul (ng) | Volume for 60ng (ul) |
#1 | 13.50 | 945.28 | 4.44 |
#2 | 9.66 | 675.94 | 6.21 |
#3 | 11.24 | 786.94 | 5.34 |
#4 | 13.64 | 955.09 | 4.40 |
#5 | 13.85 | 969.55 | 4.33 |
#6 | 8.95 | 626.85 | 6.70 |
#7 | 12.40 | 867.78 | 4.84 |
#8 | 11.29 | 790.44 | 5.31 |