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===12/11/2013=== * Lysed cells in Rui's hypotonic lysis buffer Hypotonic lysis buffer: Make 1M MgCl2 solution, filter with 0.22um filter Make 10ml 10X NP40 lysis buffer [100mM Tris.HCl pH 7.5, 100mM NaCl, 30mM MgCl2, 1% NP40, Crawford et al. PNAS 2004] Make 1ml 1X lysis buffer + 40ul protease (0.2Au/ml final) Controls introduced at first step. Positive control was 1 ul of 5.6 ng/ul E. coli gDNA. Negative control was NTC. * Digested 3 hours at 50 degC. * EtOH precipitated and resuspended in 7 ul H2O. Some cells were very large, leading to excess volume. Of these, only 7 ul was used for Nextera. * Followed standard protocol from then on: ** 7 ul sample, 2 ul 5X HMW buffer, 1 ul 1:50 Nextera. 5 min @ 55 degC. ** Protease ** Exo- ** PCR with adapters ** PCR with primers ** Ampure XP ** Size select and gel extract ** EtOH precip [[File: 2013_12_11_bulk_nxtpcr.jpg|400px]] [[File: 2013_12_11_bulk_precut1.jpg|400px]] [[File: 2013_12_11_bulk_precut2.jpg|400px]] [[File: 2013_12_11_bulk_cut1.jpg|400px]] [[File: 2013_12_11_bulk_cut2.jpg|400px]] Libraries submitted to Alan: * 7 pools ** 1: AR_bulk_25,27_Dec11 ** 2: AR_bulk_26_Dec11 ** 3: AR_bulk_28,30_Dec11 ** 4: AR_bulk_29_Dec11 ** 5: AR_bulk_31,36_Dec11 ** 6: AR_bulk_32_Dec11 ** 7: AR_bulk_37,38_Dec11
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