Alice:LabNotes/2009-1-13
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Construct Pair-end Solexa sequencing library[edit]
(Pair-end sequencing library construction) Solexa_1 adaptor and Solexa_2_PE adaptor will be used for PCR at the last step use these primers: Solexa_PCR_PE_loH & Solexa_PCR_upH (single-end sequencing library construction) Solexa_1 adaptor and Solexa_2 adaptor will be used for PCR at the last step use these primers: Solexa_PCR_up Solexa_PCR_lo
2nd PCR amplification[edit]
AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40 system setup: x4 H2O 43.2ul 172.8ul 2x Master mix 50ul 200ul dUTP(1mM) 2ul 8ul AmpF6.3(10uM) 2ul 8ul AmpR6.3(10uM) 2ul 8ul 50x SYBG I 0.4ul 1.6ul template(19ng/ul) 0.1ul 0.4ul Total 100ul 400ul
94C 3min -> 9 cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C Qiaquick column purification. 30ul * 47ng/ul
Digestion with MmeI[edit]
(dUTP 250uM,167.3ng/ul of product will use the system below) Total 20ul DNA 6ul 10X NEBuffer 4 2ul 1mM SAM(fresh) 2ul 2U/ul Mme I 8ul ddH2O 2ul 1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h
however, since the concentration I obtained from previous is 47ng/ul, therefore the following is added: DNA 14ul SAM(1mM) 4ul NEBuffer 4 2ul 37C 2hr MinElute column purify. Elute in 11ul EB.
USER digestion (1/14/09)[edit]
DNA 10ul USER 3ul total 13ul 37C 4hr
S1 nuclease digestion[edit]
10 x S1 nuclease buffer: 2ul DNA after USER digestion: 13ul S1 nuclease (10U/ul): 1ul ddH2O 4ul
37C 10mins. Minelute cloumn purify. Elute in 16ul H2O.
end repair[edit]
positive control Total 25ul 25ul DNA 15ul 2ul (obtained from Ida: 20080801_spacer_107bp) dNTP 2.5ul 2.5ul 10xendrepair buffer 2.5ul 2.5ul enzyme 0.5ul 0.5ul ATP(10mM) 5ul 5ul H2O 13ul Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 14ul H2O.
adapter ligation (1/15/09)[edit]
adaptor construction: add 10ul Solexa_2_PE_up and 10ul Solexa_2_PE_lo_noP. 10ul Solexa_2_upNH2 and 10ul Solexa_2_lo_noP. 10ul Solexa_1_upNH2 and 10ul Solexa_1_lo_noP. 95C 5mins -> 65C 30mins -> 4C.
total 30ul positive control negative control DNA 13ul 13ul ddH2O 13ul 100uM Solexa_1 adaptor 0.5ul 0.5ul 0.5ul 100uM Solexa_2_PE adaptor 0.5ul 0.5ul 0.5ul 2xQuickLiage buffer 15ul 15ul 15ul QuickLigase enzyme(NEB) 1ul 1ul 1ul extra ATP(10mM) 2.5ul 2.5ul 2.5ul Keep at room temperature (~25C) for 10 minutes. Purify with Minelute column. Elute in 20ul H2O.
PAGE selection 150-175bp. Ethanol precipitation. Elute in 15ul ddH2O. 2D gel result: File:ZhangLab 2 2009-01-16 14hr 37min.jpg the left most lane is the 25bp ladder, then cosmic adaptor ligation product, positive control, and negative control
Nick-translation (1/19/09)[edit]
set up the ligation system: Total 20ul DNA 15ul 10x ThermoPol buffer 2ul 10mM dNTP 0.4ul 1mg/ml BSA 2ul Bst polymerase(8U/ul) 1ul two tubes: product with cosmic probe and positive control
65C for 25 minutes -> keep on ice.
PCR of sequencig library[edit]
Nick-translated DNA 10ul Solexa_PCR_upH(10uM) 2ul Solexa_PCR_PE_loH(10uM) 2ul 2xiProof master mix 50ul 50x SYBG 0.8ul ddH2O 35.2ul two tubes: product with cosmic probe and positive control 98C 30sec -> 17 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold.
Purify with Qiaquick column. Elute in 30ul EB. TBE gel result: File:ZhangLab 2 2009-01-19 16hr 14min.jpg the left lane is the positive control, the middle lane with smear is the cosmic solexa sequencing product, the right lane is the 25bp ladder.
Conclusion[edit]
The solexa sequencing library preparation is successful because the PCR product had the smear at the right size. Here are few things to look out for future shot-gun library preparation. One of the most common and important cause of unsuccessful product is from the dysfunction of ATP. Since ATP provides energy for most of the biochemical reaction, it is advised to always use fresh ATP for each step. Also, it is necessary to include a positive and negative control throughout the experiment, because one needs to monitor every step in order to find the cause of any possible mistake.