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Kun:LabNotes/ExonomeSeq/2009-5-23
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==Shotgun library construction== *Start with four normalized and size-selected libraries captured from PGP1-3, NA12878 (prepared by Alice Li). The estimated concentration is ~2ng/ul each. ===PCR with dUTP=== x 4 x 4 Template: 1ul 2x EconoTaq Master Mix 50ul 800 100uM AmpF6.3NH2 0.2ul 3.2 100uM AmpR6.3NH2 0.2ul 3.2 10mM dUTP 0.6ul 9.6 50x SYBG I 0.4ul 6.4 H2O 48ul 770 94C 3min -> 9 cycles of (94C 30sec -> 58C 30sec -> 72C 20sec) -> 72C 3min -> 4C Purified every 4x50ul for each amplicon with two MinElute column. A: 23ng/ul x 20ul B: 14ng/ul x 20ul C: 14ng/ul x 20ul D: 18ng/ul x 20ul Purified each of the remaining amplicons with one DNA concentrator-5 column, eluted with 20ul EB A: 6.3ng/ul x 20ul B: 6.2ng/ul x 20ul C: 6.8ng/ul x 20ul D: 4.8ng/ul x 20ul ===USER digestion=== Qiaquick purified DNA 20ul USER enzyme 2ul 37C 1h DNA concentrator purified DNA 18ul USER enzyme 1ul 37C 1h ===S1 nuclease digestion=== 10 x S1 nuclease buffer: 3ul DNA after USER digestion: 22ul S1 nuclease (10U/ul): 2ul ddH2O 2ul 37C 15mins. Combined two tubes for each library. Purified with MinElute cloumn. Elute in 18ul H2O. A: 12ng/ul B: 7.4ng/ul C: 7.5ng/ul D: 12ng/ul ===End repair (Enzymatics End-repair Mix)=== Fragmented DNA 15ul dNTP 2.5ul 10x buffer 2.5ul Enzyme mix 1ul H2O 4ul Incubate at RT for 30 minutes Purified with Qiaquick column. Select the ~100bp fragments with 2% SizeSelect gel ===Adaptor ligation(Enzymatic T4 ligase)=== NEB DNA 34ul 2X Rapid Ligation buffer 40ul 50uM Solexa_1 adaptor 2ul 50uM Solexa_2 adaptor 2ul T4 DNA ligase(600U/ul) 2ul Keep at room temperature (~25C) for 10 minutes. Purify with MinElute columns. ===Nick translation=== DNA 8ul 10x ThermoPol buffer 1ul 10mM dNTP 0.4ul Bst Pol (8U/ul) 0.5ul 65C 10min ===PCR=== x2 x 4 DNA 5ul Solexa_PCR_up(100uM) 0.2ul Solexa_PCR_lo(100uM) 0.2ul 2x iProof master mix 50ul 50x SYBGI 0.4ul ddH2O 36ul 98C 30sec -> 8 cycles of (98C 10sec -> 65C 20 sec -> 72C 15sec) 9 cycles of (98C 10sec -> 72c 20sec) -> 72C 3min -> 4C hold. Purified 200ul amplicons with one Qiaquick column. Yield: A: 69ng/ul x 40ul B: 81ng/ul x 40ul C: 83ng/ul x 40ul D: 58ng/ul x 40ul Perform size selection on half of the ampilcons with SizeSelect gel. Quantify the selected fragments with 6% TBE gel, use the amplicon size of 160bp. Divided the concentration by 2 for the tube sent to BioGem. A: 9.7ng/ul => 92nM /2 => 46nM B: 6.2ng/ul => 58nM /2 => 29nM C: 8.5ng/ul => 81nM /2 => 40nM D: 9.1ng/ul => 86nM /2 => 82nM Perform size selection on the second half of the ampilcons with PAGE gel. ==Added another round in parallel== ===PCR with dUTP=== x 2 x 4 Template: 1.5ul 2x EconoTaq Master Mix 50ul 400 100uM AmpF6.3NH2 0.2ul 1.6 100uM AmpR6.3NH2 0.2ul 1.6 10mM dUTP 0.6ul 4.8 50x SYBG I 0.4ul 3.2 H2O 48ul 350 94C 3min -> 10 cycles of (94C 30sec -> 58C 30sec -> 72C 20sec) -> 72C 3min -> 4C Purified every 200ul for each amplicon with one Qiaquick column. A: 54ng/ul x 28ul B: 45ng/ul x 28ul C: 40ng/ul x 28ul D: 38ng/ul x 28ul ===USER digestion=== Qiaquick purified DNA 18ul USER enzyme 2ul 37C 1h ===S1 nuclease digestion=== 10 x S1 nuclease buffer: 3ul DNA after USER digestion: 20ul S1 nuclease (10U/ul): 2ul ddH2O 4ul 37C 10mins. Purified with MinElute cloumn. Elute in 18ul H2O. A: 11ng/ul B: 9ng/ul C: 7ng/ul D: 5ng/ul Hold the samples at this step.
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