Kun:LabNotes/ASE/2007-12-31: Difference between revisions

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==Exp.1 Make gDNA-free cDNA from GM20431, GC1EP and Hues6==
==Exp.1 Make gDNA-free cDNA from GM20431, GC1EP and Hues6==
===RNA clean-up===
                   GM20431(06/04/07)  GC1EP(06/14/07) Hues6(05/14/07)
                   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
     total RNA      1.25ug/ul x8ul    645ng/ul x 15ul  270ng/ul x 25ul
Line 7: Line 8:
     RNase Inhibitor  0.5ul              0.5ul            0.5ul
     RNase Inhibitor  0.5ul              0.5ul            0.5ul
     37C 30min -> 75C 10min -> Cleanup with RNeasy columns
     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===
                    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
==Exp.2 Also process RNAs from Hybrid1, Hybrid2, BJ Fibroblast==
==Exp.2 Also process RNAs from Hybrid1, Hybrid2, BJ Fibroblast==
                       Hybrid1            Hybrid2        BJ Fibroblast
                       Hybrid1            Hybrid2        BJ Fibroblast

Revision as of 00:26, 1 January 2008

Exp.1 Make gDNA-free cDNA from GM20431, GC1EP and Hues6

RNA clean-up

                  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

                   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

Exp.2 Also process RNAs from Hybrid1, Hybrid2, BJ Fibroblast

                     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