Kun:LabNotes/ASE/2007-12-31
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Exp.1 Make gDNA-free cDNA from GM20431, GC1EP and Hues6[edit]
RNA clean-up[edit]
GM20431(06/04/07) GC1EP(06/14/07) Hues6(05/14/07) total RNA 1.25ug/ul x8ul 645ng/ul x 15ul 270ng/ul x 25ul 10x buffer 3ul 3ul 3ul H2O (DEPC) 17ul 10ul 0ul DNase I(2u/ul) 2ul 2ul 2ul RNase Inhibitor 0.5ul 0.5ul 0.5ul 37C 30min -> 75C 10min -> Cleanup with RNeasy columns
Yield:
- GM20431: 338ng/ul x 30ul
- GC1EP: 265ng/ul x 30ul
- Hues6: 36ng/ul x 30ul
First strand synthesis[edit]
GM20431 GC1EP Hues6 RNA 25ul 25ul 25ul 10mM dNTP 3ul 3ul 3ul 50uM Oligo dT 3ul 3ul 3ul 65C 5min -> place on ice for 1min Add 10x RT buffer 6ul 6ul 6ul 25mM MgCl2 12ul 12ul 12ul 0.1M DTT 6ul 6ul 6ul RNase OUT(40U/ul) 2ul 2ul 2ul SS III RT(200U/ul) 3ul 3ul 1ul 50C 50min -> 85C 5min
Second strand synthesis[edit]
Cleanup the first-strand reactions with G-25 columns.
To the ~60ul reactions, add H2O (DEPC) 22ul 10ul NEBuffer 2 10ul 10mM dNTP 3ul DNA Pol I(10U/ul) 4ul RNase H (2U/ul) 1ul Mix well, incubate at 16C for 2h, purified with MinElute columns.
Yield:
- GM20431: 471ng/ul x 15ul -> add 35ul EB -> 122ng/ul x 45ul
- GC1EP: 320ng/ul x 15ul -> add 20ul EB -> 114ng/ul x 30ul
- Hues6: 53ng/ul x 10ul
Exp.2 Also process RNAs from Hybrid1, Hybrid2, BJ Fibroblast[edit]
Hybrid1 Hybrid2 BJ Fibroblast total RNA 1.9ug/ul x8ul 570ng/ul x 20ul 72ng/ul x 52ul 10x buffer 3ul 3ul 6ul H2O (DEPC) 17ul 5ul 0ul DNase I(2u/ul) 2ul 2ul 2ul RNase Inhibitor 0.5ul 0.5ul 0.5ul 37C 30min -> 75C 10min -> Cleanup with RNeasy columns
Yield:
- H1 hybrid: 59ng/ul x 30ul
- H2 hybrid: 41ng/ul x 30ul
- BJ Fibroblast: 26ng/ul x 30ul
First strand synthesis[edit]
GM20431 GC1EP Hues6 RNA 25ul 25ul 25ul 10mM dNTP 3ul 3ul 3ul 50uM Oligo dT 3ul 3ul 3ul 65C 5min -> place on ice for 1min Add 10x RT buffer 6ul 6ul 6ul 25mM MgCl2 12ul 12ul 12ul 0.1M DTT 6ul 6ul 6ul RNase OUT(40U/ul) 2ul 2ul 2ul SS III RT(200U/ul) 3ul 3ul 1ul 50C 50min -> 85C 5min
Second strand synthesis[edit]
Cleanup the first-strand reactions with G-25 columns.
To the ~60ul reactions, add H2O (DEPC) 22ul 10ul NEBuffer 2 10ul 10mM dNTP 3ul DNA Pol I(10U/ul) 4ul RNase H (2U/ul) 1ul Mix well, incubate at 16C for 2h, purified with MinElute columns.
Yield:
- H1: 123ng/ul
- H2: 72ng/ul
- BJ: 62ng/ul