Kun:LabNotes/ExonomeSeq/2008-1-14
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Making sequencing libraries from the 250bp bands purified from 01/09/08 exp[edit]
DNA synthesis with random hexamers[edit]
N6 DNA 20ul 10X Klenow buffer 3ul 1mM N6 0.6ul 10mM dNTP 1.2ul H2O 2.2ul
94C 2min -> 16C 1min -> add 1.5ul 20U/ul Klenow exo- -> 16C 10min -> 37C 5min -> 94C 1min -> 16C 1min -> add 1.5ul 20U/ul Klenow exo- -> 16C 10min -> 37C 10min -> purified with MinElute columns.
USER enzyme digestion[edit]
Add 2ul USER enzyme to 20ul purified DNA in dH2O -> 37C 1h -> purified with MinElute columns.
Ligation to Solexa adaptors[edit]
- Mix 100ul of Solexa_1_up and Solexa_1_lo_noP adaptors (200uM) with 200ul 1X PCR buffer, the final concentration is 50uM.
- Mix 100ul of Solexa_2_up and Solexa_2_lo_noP adaptors (200uM) with 200ul 1X PCR buffer, the final concentration is 50uM.
- 95C 5min -> turn of the program, let the tube slowly cold down in ~10 minute -> transfer on ice.
N6 primed USER digested DNA 13ul 22ul 50uM Solexa_1 1ul 1ul 50uM Solexa_2 1ul 1ul 2X Quick ligation buffer 25ul 25ul Quick ligase 1ul 1ul H2O 9ul 0ul mix well -> room temperature for 20min -> 75C 10min Run 20ul in 4% clonewell gel, collect the fraction of 100~150bp.
Purify the remaining 20ul reactions with MinElute columns.
Nick translation[edit]
DNA 15ul 10X ThermoPol Buffer 2ul 10mM dNTP 0.4ul 1mg/ml BSA 2ul 8U/ul Bst pol 1ul 65C 25min -> place on ice
PCR enrichment of ligated DNA[edit]
Nick-translated DNA 5ul 2X iProof master mix 25ul 100uM Solexa_PCR_up 0.25ul 100uM Solexa_PCR_lo 0.25ul H2O 19.3ul 50X SYBG I 0.2ul 98C 30sec -> 18 cycles of (98C 10sec -> 68C 30sec) -> 72C 3min ->15C hold File:2008-01-17-E55kv1-SolLib-PCR.png