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==Sample preparation== Name DNA(20ng) tRNA carrier(480ng) RT-PCR H2O ---------------------------------------------------- 90m-3 45.8 4.8 49.4 10h-3 2.0 4.8 93.2 90m-4 40.0 4.8 55.2 10h-4 2.3 4.8 92.9 *Only use about 20ng DNA for starting amount (10ng is the total available amount for the sample Sam072710-90m-3) ===EtOH purification of MDA amplicons=== *Add 4.8uL tRNA carrier(100ng/uL) into MDA amplicon. *Add H2O to make total valume to 100uL. *Add 2.5X volume 100% EtOH (250uL) + 0.1X volume 3M NaOAc (10uL) + 1.5uL Glycoblue -> incubate at -80C for 25min. *Centrifuge at 10,000xg, 4C for 25min -> resuspend the pellet with 750uL 75% EtOH. *Centrifuge at 10,000xg, 4C for 5min. Elute the pellet in 15uL RT-PCR grade H2O. ===Tagmentation reaction=== *Set up tagmentation reaction on cold block: 1rxn DNA(10ng)+ H2O 15.0 5X Nextera Rxn buf.(LMW) 4.0 Nextera enzyme 1.0 ------------------------------ 20.0 uL *Briefly vortexing and centrifuging. Incubate in thermocycler (using instant incubation) at 55C for 5min. *During the incubation, setup the tube and buffer of Zymo DNA cleaning kit ===Zymo DNA purification=== *Pre-load 100uL binding buf. in 1.5mL-tubes. Add 20uL reaction product from last step. *Briefly vortexing and centrifuging. *Transfer the mixture (120uL) onto a Zymo-spin column in a collection tube. *Centrifuge at 10,000xg for 1min. Discard flowthrough. *Add 250uL washing buf. onto the column. Centrifuge at 10,000xg for 1min. Discard flow-through. *Repeat the last step again. *Perform an additional centrifuge at 10,000xg for 1min (to eliminate any residue). *Add 11uL pre-warmed H2O onto the column, transfer the column onto a 1.5mL-tube. Incubate at RT for 2min. *Centrifuge at 10,000xg for 1min. The elute DNA is about 10~11uL. Only use 5uL for the library amplification. (Label and store the other 5uL product in -20C) ===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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