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==Procedures== ===EtOH purification of MDA amplicons=== **Add 4.5uL 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(50ng)+ 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 flowthrough. *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=== *Set up the reaction on cold block in PCR tubes: 1rxn RT-PCR grade H2O 17.0 DNA lib (from last step) 5.0 2X Nextera PCR buf. 25.0 50X Nextera primier cocktail 1.0 - Illumina-compatible 50X Nextera adaptor 2 1.0 - could be stwitch to barcoded library (optional) Netera PCR enzyme 1.0 --------------------------------- 50.0 uL *Briefly vortexing and centrifuging. *Program "Nextera": 72C 3min-> 95C 30sec -> (95C 10sec -> 62C 30sec -> 72C 3min)x12 cycle -> 4C forever *NOTE: the result should be about 500ng amplified DNA (Zymo manual).
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