Alice:LabNotes/2010-1-21
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MmeI digestion on CViB sequencing libraries[edit]
- Previous library construction protocol can be found under labnotes 1-9-10
- size selection protocol can found under labnotes 1-15-10
setup: DNA(15nM) 10ul 10x buffer 4 2ul 1mM SAM 2ul MmeI(2U/ul) 2ul 37C 1hr, 80C 20min TBE gel result: File:ZhangLab 2 2010-01-23 18hr 01min.jpg
RE-amplify CViB sets for Covaris shearing[edit]
- previously captured libraries on CViB and PGP7 sets
- the protocol used for the previous steps can be found under labnotes 12-18-09 and 1-5-10 (for CViB sets) and 8-31-09 (for PGP7)
- for PGP7, capture products with Jan06 and Jan07 probes were disregarded
PGP7 1 2 3 concnetration(ng/ul): 4.4ng/ul 4.5ng/ul 2.9ng/ul volume needed (ul): 2.5ul 1.2ul 2.75ul
CViB-ab: (a)1ul + (b)1ul CViB-cde: (c)1ul + (d)1.6ul + (e)1.5ul
PCR x8(for each set) Template: ~0.25-1ul (total of 2ul for CViB-ab, 4.1ul for CViB-cde, 6.45ul for PGP7) 2x Phusion master mix: 50ul 100uM AmpF6.3NH2 0.2ul 100uM AmpR6.3NH2 0.2ul 50X SYBG I: 0.4ul H2O 50ul 98C 30sec -> (98C 10sec -> 58C 20sec -> 72C 20sec) x 6 -> 72C 3min -> 4C hold. (11 cycles for PGP7) Qiaquick column purification and elute in 40ul ddH2O. Nanodrop: CViB-ab: 13.7ng/ul PGP7: 46.7ng/ul CViB-cde: 58.6ng/ul