Jie:LabNotes/CpgSeq/2009-9-8
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shotgun library construction of PGP samples captured by cpg97k
The captured DNA of samples were sent to Billy for shearing.refer to LabNotes on [1]
digest with MmeI
x7 Total 160ul DNA 100ul 10X NEBuffer 4 16ul 112 1mM SAM(fresh) 32ul 224 2U/ul Mme I 8ul 56 ddH2O 4ul 28 1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h MinElute column purify. Elute in 20ul EB.
endrepair with enzymatic end-repair kit
PGP samples x7 20 ul DNA 3 ul 10X End-Repair Buffer 21 3 ul dNTP Mix 21 4 ul End-Repair Enzyme Mix 28 30 ul Total reaction volume
DF-iPS x2 100 ul DNA 13 ul 10X End-Repair Buffer 26 13 ul dNTP Mix 26 4 ul End-Repair Enzyme Mix 8 130 ul Total reaction volume
Incubate at room temperature for 30 minutes. Purify with minelute column. Elute in 20ul ddH2O. E-gel size selection:
P09_2,P09_3,DF30k and DF97K: select 100bp; The others: select 150bp..
A tail addition
x9 Blunt-ended DNA 30ul 30 each 10X Klenow buffer 4ul 36 1mM dATP 6ul 54 Klenow fragment (exo-)10U/ul 2ul 18 37C 30min, purified with MinElute columns, eluted with 12ul EB.
adaptor ligation
x12 DNA 10ul 2x QuickLigase buffer (enzymatic) 15ul 180 20uM Adaptor oligo mix 3ul 36 T4 DNA QuickLigase (enzymatic) 2ul 24 Incubate at RT for 15 minutes. Purified with Qiaquick columns, eluted with 30ul EB.
PCR
Template 15ul 2x iProof mix 50ul Solexa_PCR_up (10uM) 4ul Solexa_PCR_lo_PE (10uM) 4ul H2O 27ul 50X SYBG I 0.2ul 98C 30sec -> 5 cycles of (98C 10sec -> 65C 20sec -> 72C 15 sec) -> 7 cycles of (98C 10sec -> 72C 15 sec)-> 72C 3min -> 15C hold.
quantification of shotgun library with qPCR
I dilute the PhiX lib(10nM) to 1nM, 0.5nM, 0.25nM, 0.05nM. I dilute the Parkinson's lib with 1:10 ratio. Syb_FP5: ATGATACGGCGACCACCGAG Syb_RP7: CAAGCAGAAGACGGCATACGAG x 14 Template 1ul 2x iProof mix 25ul 350 syb_RP7 (100uM) 0.2ul Syb_FP5 (100uM) 0.2ul H2O 24ul 50X SYBG I 0.2ul
98C 30sec -> 10 cycles of (98C 10sec -> 65C 20sec -> 72C 15 sec) ->72C 3min -> 15C hold.
No | sample | sample concentration |
Par_1 | 5028 | 12.2nM |
Par_2 | 4735 | 10.2nM |
Par_3 | 4789 | 9.3nM |
Par_4 | 5171 | 6.6nM |
Par_5 | 1818 | 6.0nM |
Par_6 | 1947 | 7.8nM |
Par_7 | 1741 | 6.9nM |
Par_8 | 4977 | 9.6nM |
Par_9 | 4879 | 5.5nM |
Par_10 | 4526 | 6.6nM |