Jie:LabNotes/CpgSeq/2009-8-5
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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.
After A tail, I did size selection for No7 and positive control, then do the adaptor ligation.
After adaptor ligation, i goes to PCR directly without size selection again.
However, I can only see the positive control product only.
So I skipped the size selection before adaptor ligation for No 1~6, and do the size selection after adaptor ligation. However, the expected band after PCR is so weak that I am not sure the targets were successfully amplified.
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
File:20090721 shotgun libr par7 positive control.jpg20090721_shotgun_libr_par7_positive control
File:20090721 shotgun libr par1~6.jpg20090721_shotgun_libr_par1~6
redo the shotgun library starting from PCR amplification with AmpF6.3NH2/AmpR6.3NH2 (2009-07-22)
I did the PCR reamplification with Amp6.3NH2 primers. 400ul for each sample. reaction system x4 x17 H2O 50ul 200ul 3400 2x Mastermix 50ul 200ul 3400 AmpF6.3NH2(100uM) 0.2ul 0.8ul 13.6 AmpR6.3NH2(100uM) 0.2ul 0.8ul 13.6 50x SYBG I 0.4ul 1.6ul 27.2 template 1ul/each for purified cpg97k Total 100ul 800ul 94C 2min -> (94C 45S -> 55C 45S -> 72C 45S) x 8 ->72C 3 min -> 15C hold.
I did the PCR reamplification with Amp6.3 primers with Parkinson's sample 1 and 10 with the same PCR condition.