Alice:LabNotes/2009-3-13
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Objective
1.Construct single-end solexa sequencing library using PCR-product of probes captured with sheared, short DNA PGP1. 2.Compare the MmeI digestion efficiency between established capture method and shaking.
Material
First attempt: Obtained two tubes from Dr. Zhang: (1) PGP1-CTRL 56ng/ul; (2) PGP1-shaked 27ng/ul Dilute tube (1) 10X by adding 9ul of H2O to 1ul of DNA sample. For tube (2), only very tiny amount of DNA was left in the tube. Took out around 1ul of DNA and add 9ul of H2O. Nanodrop result gives out final concentration of 6ng/ul. (1) PGP1-shaked: 6ng/ul (2) PGP1-CTRL: 5.6ng/ul
Second attempt (3-16-09): (1) PGP1-shaked: 12ng/ul (2) PGP1-CTRL: 5.6ng/ul
Procedure
Reagent setup: (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
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 1ul 4ul Total 100ul 400ul 94C 3min -> 10 cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
Qiaquick column purification. tube 1 (shaked): 30ul * 257.1ng/ul tube 2 (CTRL): 30ul * 194.6ng/ul
Second attempt (3-16-09): 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 (tube1 / tube2) 1ul/0.5ul 4ul/2ul Total 100ul 400ul 94C 3min -> 10 cycles of (94C 45sec -> 55C 45sec -> 72C 45sec) -> 72C 3min -> 4C
Qiaquick column purification. tube 1 (shaked): 30ul * 87.2ng/ul tube 2 (CTRL): 30ul * 75.9ng/ul
Digestion with MmeI
(dUTP 250uM,167.3ng/ul of product will use 6ul of DNA along with 2ul of 1mM SAM and 8ul of 2U/ul MmeI) First attempt: Total 20ul tube 1(257.1ng/ul) 4ul tube 2 (194.6ng/ul) 6ul 10X NEBuffer 4 2ul 10X NEBuffer 4 2ul 1mM SAM(fresh) 2ul 1mM SAM(fresh) 2ul 2U/ul Mme I 8ul 2U/ul Mme I 8ul ddH2O 4ul ddH2O 2ul
Second attempt: Total 40ul DNA 13ul 10X NEBuffer 4 4ul 1mM SAM(fresh) 6ul 2U/ul Mme I 8ul ddH2O 9ul 1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h
MinElute column purify. Elute in 11ul EB. TBE gel result: File:ZhangLab 2 2009-03-14 16hr 19min - TBE.jpg TBU gel result: File:ZhangLab 2 2009-03-17 10hr 59min-TBU.jpg
USER digestion (1/14/09)
DNA 10ul USER 3ul total 13ul 37C 4hr
S1 nuclease digestion
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
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)
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 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. TBE gel result: File:ZhangLab 2 2009-03-18 10hr 45min.jpg
Nick-translation (1/19/09)
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
Nick-translated DNA 10ul Solexa_PCR_up(100uM) 0.2ul Solexa_PCR_lo(100uM) 0.2ul 2xiProof master mix 50ul 50x SYBG 0.8ul ddH2O 35.2ul two tubes: product with cosmic probe and positive control 98C 30sec -> 10 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold. File:3-18-09 PCR of solexa library.jpg red line: control blue line: PGP1-shaked Green line: PGP1-CTRL
Purify with Qiaquick column. Elute in 30ul EB. TBE gel result: File:ZhangLab 2 2009-03-18 15hr 20min.jpg
Result
There is no significant amount of difference between the Mme1 digestion of CTRL set and shaked DNA set. Therefore we cannot say that one method is preferred over another. Also, the gel image of Mme1 digestion showed 3 bands that suggests incomplete digestion. A gel selection might be helpful after Mme1 digestion.
Discussion
It is very crucial to use TBE gel to do size selection after adapter ligation step. It is because after adapter ligation, the double stranded DNA will ligate with only one strand of the double stranded solexa adapter, and leaving a gap between the other strand of the adapter and the captured probe. This is also the reason why a nick translation step is necessary before PCR amplification, because it is used to seal up the gap. A TBU gel will denature the DNA and causing the unligated strand of adapter to disassociated from the adapter and the captured probe, thus giving a undesirable product and the experiment fails.