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Jeff:LabNotes/Microbiome/2012-8-16
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== '''Library Construction: Microwell Amplicons ''' == *MDA seems to have worked based on fluorescence. *Extracted 2 of each sample in 1 uL water *Nuclei had by far the greatest fluorescence in rtMDA *HMW buffer in tagmentation *Use Pol1 +ALS/NS treatment '''Pol 1 treatment''' *Start with 1 uL DNA *Add 1.5 uL ALS, 3min *Put on ice and add 1.5 uL NS Template 4 uL 10x buffer 2 uL dNTP 1 uL N6(unmodified) 5 uL water 7 uL Pol 1 1 uL ---------------------------------- Total 20 uL *37 C for 1 hr, 65C for 10 min *Standard EtOH (Add 200 ng tRNA carrier) and elute in 7 uL water. The tRNA was newly treated with DNAse '''Tagmentation''' DNA in water 7 uL HMW buffer 2 uL, 1:50 diluted enzyme 1 uL ---------------------- Total 10 uL *55C for 5 min '''Protease treatment''' *Add 1 uL 1:100 diluted protease *10 min 50C, 20 min 70C '''Klenow''' *Add 1 uL exo- klenow and 1 uL dNTP to each *37C for 15 min '''1st PCR round''' DNA 13 uL KAPA Robust mix 25 uL Primer cocktail 1 uL 1 uM barcode (25-37) 1 uL water 9.6 uL 50x SYBR .4 uL -------------------------- Total 50 uL *95 C 30s (95 C 10s, 62C 15s, 72 C 2 min)x20 '''Bead purfication''' *Add 50 uL beads to each, mix, and let sit at RT for 8min *Transfer tubes to magnetic rack and let sit for 5 min *Remove supernatant *2x 80% EtOH wash *Sit for 15 min *Elute in 23 uL Qiagen buffer '''Results''' *Tagmentation worked and got good results *Some amplified better than others *Save libraries for validation [[File:8-16-12 pcr.jpeg|400px]] [[File:8-16-12 gel_2.jpeg|400px]] [[File:8-16-12 gel.jpeg|400px]]
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