Jie:LabNotes/CpgSeq/2009-8-5: Difference between revisions
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>Jie deng (New page: ==shotgun library construction of Parkinson's samples== ====endrepair with epicentre kit==== 100 ul DNA 15 ul 10X End-Repair Buffer 15 ul dNT...) |
>Jie deng No edit summary |
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Klenow fragment (exo-) 1ul 8 | Klenow fragment (exo-) 1ul 8 | ||
37C 30min, purified with MinElute columns, eluted with 20ul EB. | 37C 30min, purified with MinElute columns, eluted with 20ul EB. | ||
====adaptor ligation==== | ====adaptor ligation==== | ||
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Solexa_PCR_lo_PE (10uM) 4ul | Solexa_PCR_lo_PE (10uM) 4ul | ||
H2O 30ul | H2O 30ul | ||
50X SYBG I 0.2ul | 50X SYBG I 0.2ul | ||
Revision as of 18:46, 4 August 2009
shotgun library construction of Parkinson's samples
endrepair with epicentre kit
100 ul DNA 15 ul 10X End-Repair Buffer 15 ul dNTP Mix 15 ul ATP 3 ul End-Repair Enzyme Mix 150 ul Total reaction volume Incubate at room temperature for 45 minutes. Purify with minelute column.
A tail addition
x8 Blunt-ended DNA 10ul 80 10X Klenow buffer 1.5ul 12 1mM dATP 3ul 24 Klenow fragment (exo-) 1ul 8 37C 30min, purified with MinElute columns, eluted with 20ul EB.
adaptor ligation
DNA 20ul 10x QuickLigase buffer (enzymatic) 3ul 20uM Adaptor oligo mix 3ul T4 DNA QuickLigase (enzymatic) 2ul Incubate at RT for 15 minutes. Purified with Qiaquick columns, eluted with 12ul EB
PCR
Template 15ul 2x iProof mix 50ul Solexa_PCR_up (10uM) 4ul Solexa_PCR_lo_PE (10uM) 4ul H2O 30ul 50X SYBG I 0.2ul