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===Library enrichment by PCR amplification=== *In order to use the libraries for cloning sequencing, we have to switch to the Phusion enzyme instead. **The Phusion enzyme provide blunt end PCR products which is suitable for blunting cloning. **We also need to add Isotherm (Epicentre) enzyme to repair the gaps left by transposon taggmentation. *In order to test the new amplification recipe, I only tried two of four samples with barcoded(Index) primer **90m-4(IDX1) and 10hr-4(IDX2) *Set up the qPCR reaction (50uL) on cold block in PCR tubes: 1rxn RT-PCR grade H2O 17.1 DNA lib (from last step) 5.0 2X Phusion master mix 25.0 50X Nextera primier cocktail 1.0 - Illumina-compatible 50X Nextera adaptor 2 1.0 - Using IDX1 and IDX2 50X SYBR I 0.4 Isothermo(Epicentre) 0.5 --------------------------------- 50.0 uL *Briefly vortexing and centrifuging. **65C 3min -> 98C 30sec -> (98C 5sec -> 65C 15sec -> 72C 20sec)x15 cycle -> 72C 3min -> 4C forever''' **'''NOTE1.: This program is adapted from Dr.Zhang's protocol 0805'10. The temperature setting is different from original Nextera protocol. **'''NOTE2.: The 65C 3min is for Isothermo enzyme. The 98C 30sec is for Phusion (Hot-start) enzyme'''. *Stop the reaction before the amplification reach saturation. **Sample 90m-4 stopped at 16cycle **Sample 10h-4 stopped at 8cycle
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