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==Continuation of Library Construction== [[http://genome-tech.ucsd.edu/LabNotes/index.php/AlanFung:LabNotes/CTCF/2010-2-8]] ===MET_Y_adapter Purification=== *Perform ethanol precipitation to purify adapter *add 10ul 3M NaoAC and 250ul 100% EtoH and 1ul glycoblue mix and freeze in -80C for 20M *spin at 10,000rpm for 20 mins at 4C *discard supernatant *Add 500ul of 75% etoh and spin at 10,000rpm at 4C *discard supernatant and let dry in hood for 10mins *resolve in 100ul ddh2o ===TBE gel verification=== [[File:ZhangLab_2 2010-02-22 14hr 08min.jpg]] *Band with very large size bp showed up adapter should be in there, move on with adapter ligation ===Quantify using qubit=== *Mix 1ul dye with 199ul buffer *Mix 2ul adapter with 98ul diluted dye *mix and incubate for 2 mins Results: 4.24ng/ul *100ul = 424ng ===Ligation. Blunt-end and TA ligations use the same protocol but slightly different adaptor sequences=== * Set up ligation reaction. Note that the ATP in the Quick Ligase buffer hydrolyzes very quickly after several rounds of freeze/thaw cycles, so itβs a good idea to make small aliquots of a fresh tube of Quick Ligase Buffer, and use one small aliquot each time. {| {{table}} | ||ul |- | A-tailed input DNA||50 |- | 100ng MET_Y_adapter(4.24ng/ul)||23 |- | 5X Quick Ligase Buffer||20 |- | Quick T4 DNA Ligase||3 |- | ddh2o||5 | |} Incubate at room temperature for 15 minutes, purified with MinElute columns, eluted with 30ul EB. *Add 5X Volume PB w/ sample mix *spin and discard at 14,000rpm for 1min *add 750ul wash buffer *spin and discard x2 *Add 32ul eb buffer wait for 5 mins and spin to collect tube *Aliquot 28ul for CT conversion and 2ul for qPCR with PhiX
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