Alice:LabNotes/2010-1-21: Difference between revisions
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>Zsakura2 |
>Zsakura2 |
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concnetration(ng/ul): 4.4ng/ul 4.5ng/ul 2.9ng/ul | concnetration(ng/ul): 4.4ng/ul 4.5ng/ul 2.9ng/ul | ||
volume needed (ul): 5ul 2.4ul 5.5ul | volume needed (ul): 5ul 2.4ul 5.5ul | ||
------------------------------------------------------------------- | |||
total (ng): 22ng 11ng 16ng | total (ng): 22ng 11ng 16ng | ||
CViB-ab: | |||
(a)1.5ul + (b)1.5ul | |||
CViB-cde: | |||
(c)1ul + (d)1.6ul + (e)1.5ul | |||
PCR | PCR | ||
Template: | Template: 0.25ul | ||
2x Phusion master mix: | 2x Phusion master mix: 50ul | ||
100uM AmpF6.3NH2 | 100uM AmpF6.3NH2 0.2ul | ||
100uM AmpR6.3NH2 | 100uM AmpR6.3NH2 0.2ul | ||
50X SYBG I: | 50X SYBG I: 0.4ul | ||
H2O | H2O 50ul | ||
98C 30sec -> (98C 10sec -> 58C 20sec -> 72C 20sec) x 6 -> 72C 3min -> 4C hold. | 98C 30sec -> (98C 10sec -> 58C 20sec -> 72C 20sec) x 6 -> 72C 3min -> 4C hold. |
Revision as of 22:18, 21 January 2010
MmeI digestion on CViB sequencing libraries
- 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
RE-amplify CViB sets for Covaris shearing
- 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): 5ul 2.4ul 5.5ul ------------------------------------------------------------------- total (ng): 22ng 11ng 16ng
CViB-ab: (a)1.5ul + (b)1.5ul CViB-cde: (c)1ul + (d)1.6ul + (e)1.5ul
PCR Template: 0.25ul 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.