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