Noi/NOTES/2012-8-1: Difference between revisions

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[[File:ZhangLab_2 2012-08-02 18hr 23min-PAGE-SS.jpg| 400px]]

Revision as of 01:39, 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

File:ZhangLab 2 2012-08-02 18hr 23min-PAGE-SS.jpg