Kun:LabNotes/ASE/2008-1-11
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Exp. 1 RNA clean-up & RT on GC1F, Hues30[edit]
Received GC1F from Jay Lee today. The shipping was delayed but there was still dry ice in the box, so RNA should be fine.
RNA clean-up[edit]
Clean up 20ul GC1F total RNA (0.5ug/ul) and 10ul Hues30 total RNA (0.8ug/ul) with RNeasy Plus columns. Yield:
- GC1F: 200ng/ul x 30ul
- Hues30: 60ng/ul x 30ul
Also did DNase I digestion on GC1F total RNA. 8ul 0.5ug/ul total RNA + 1ul 10X DNase I buffer + 1ul DNase I (RNase free), 5U/ul 37C 30min -> 80C 20min.
Reverse transcription[edit]
GC1F(0.5ug/ul) GC1F(0.2ug/ul) GC1F(DNase) Hues30(60ng/ul) RNA 4ul 25ul 5ul 25ul 10mM dNTP 1ul 3ul 1ul 3ul 50uM d(T) 1ul 3ul 1ul 3ul H2O 4ul 0ul 3ul 0ul 65C 5min -> place on ice for > 1min Add 10X RT buffer 2ul 6ul 2ul 6ul 25mM MgCl2 4ul 12ul 4ul 12ul 0.1M DTT 2ul 6ul 2ul 6ul RNase OUT 1ul 2ul 1ul 2ul SS III 1ul 3ul 1ul 3ul 37C 10min -> 50C 50min -> 85C 5min
Second strand synthesis[edit]
Clean up the first strand reactions with G-25 columns.
to the 20/60ul reactions, add H2O (DEPC) 11ul/22ul 10X NEBuffer 2 4ul/10ul 10mM dNTP 2ul/3ul DNA Pol I 2ul/4ul RNase H 1ul/1ul Mix well, incubate at 16C for 2h, purified with MinElute columns. Yield: 57ng/ul 167ng/ul 44ng/ul 44ng/ul
Exp.2 PCR on the eSNP84k capturing reactions started on 01/08/08[edit]
x 1 x 14 template: 10ul each JumpStart buffer: 9ul 126ul 10mM dNTP: 2ul 28ul 100uM AmpF2Sol: 0.4ul 5.6ul 100uM AmpRSol: 0.4ul 5.6ul 50X SYBG I: 0.8ul 11.2ul JumpStart: 2ul 28ul H2O: 75ul 1050ul
94C 3min -> 8 cycles of (94C 45sec -> 56C 2min -> 72C 1min) -> 10 cycles of (94C 45 sec -> 72C 1min) -> 72C 3min -> 15C hold.