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==single-end Shotgun sequencing library construction== ===PCR with dUTP=== x 4 x4 Template: 0.25ul 1ul 2x EconoTaq Master Mix 50ul 200ul 100uM AmpF6.3NH2 0.2ul 0.8ul 100uM AmpR6.3NH2 0.2ul 0.8ul 1mM dUTP 4ul 16ul 50x SYBG I 0.4ul 1.6ul H2O 50ul 200ul 94C 3min -> 10 cycles of (94C 30sec -> 58C 30sec -> 72C 20sec) -> 72C 3min -> 4C Purified the amplicon with one Qiaquick column each e-1: 14.2ng/ul x 30ul e-2: 31.1ng/ul x 30ul f-1: 13.3ng/ul x 30ul f-2: 33.6ng/ul x 30ul [[Image:ZhangLab_2 2009-06-05 11hr 19min.jpg]] ===USER digestion=== Qiaquick purified DNA 30ul USER enzyme 2ul 37C 1h ===S1 nuclease digestion=== 10 x S1 nuclease buffer: 4ul DNA after USER digestion: 32ul S1 nuclease (10U/ul): 1ul ddH2O 3ul 37C 15mins Purified with MinElute column. Elute in 18ul H2O. Nanodrop: e-1:5ng/ul e-2:7.1ng/ul f-1:6.8ng/ul f-2:10.1ng/ul ===End repair (epicentre)=== pos. control Fragmented DNA 15ul Spacer 107bp 2ul + H2O 13ul dNTP 2.5ul 10x buffer 2.5ul enzyme 0.5ul ATP 5ul Incubate at RT for 40 minutes Purified with minelute column and elute in 20ul Select the ~100bp fragments with Egel (only the seq. library samples, positive control not included). ===Adaptor ligation=== pos. control neg control DNA 16ul 20ul H2O 16ul 2X Rapid Ligation buffer 18ul 22ul 18ul 100uM Solexa_1 adaptor 0.5ul 0.5ul 0.5ul 100uM Solexa_2 adaptor 0.5ul 0.5ul 0.5ul QuickLigase(400U/ul) 1ul 1ul 1ul Keep at room temperature (~25C) for 10 minutes. Purify with MinElute columns and elute in 16ul EB. performed one round of size selection on positive control tube using 2% Size Select E-gel. The total amount obtained is 16ul. ===Nick translation=== DNA 15ul 10x ThermoPol buffer 2ul 10mM dNTP 0.4ul BSA 2ul Bst Pol (8U/ul) 1ul 65C 20min TBE gel size selection at 150-170bp ===PCR=== x 6 DNA 10ul Solexa_PCR_up(100uM) 0.2ul Solexa_PCR_lo(100uM) 0.2ul 2x iProof master mix 50ul 50x SYBGI 0.4ul ddH2O 40ul 98C 30sec -> 6 cycles of (98C 10sec -> 65C 20 sec -> 72C 15sec)-> 7 cycles of (98C 10 sec -> 72C 20sec) -> 72C 3min -> 4C hold. [[Image:ZhangLab_2 2009-06-05 16hr 02min.jpg]] x 6 DNA 10ul Solexa_PCR_up(100uM) 0.2ul Solexa_PCR_lo(100uM) 0.2ul 2x iProof master mix 50ul 50x SYBGI 0.4ul ddH2O 40ul 98C 30sec -> 6 cycles of (98C 10sec -> 65C 20 sec -> 72C 15sec)-> 6 cycles of (98C 10 sec -> 72C 20sec) -> 72C 3min -> 4C hold. [[Image:ZhangLab_2 2009-06-07 19hr 24min.jpg]] Qiaquick column purification and elute in 40ul of EB Nanodrop: e-1: 24.8ng/ul e-2: 34.4ng/ul f-1: 30.0ng/ul f-2: 34.7ng/ul ===result=== gel quantification result: e-1: PGP5-1-060809 (7.43ng/ul = 62.5nM) e-2: PGP5-2-060809 (7.81ng/ul = 65.7nM) f-1: PGP7-1-060809 (8.98ng/ul = 75.5nM) f-2: PGP7-2-060809 (9.47ng/ul = 79.7nM) send out 10ul of each for sequencing (for lane 4-7) and also 10ul of f-2: PGP7-2-060809 for sequencing (for lane 8)
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