Kun:LabNotes/ASE/2007-11-19

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Exp. 1 Making ds-cDNA for the total RNA from the hybrid lines[edit]

Templates:

  • Hybrid 1 total RNA: 1.9ug/ul
  • Hybrid 2 total RNA: 0.44ug/ul
  • BJ fibroblast total RNA: 110ng/ul

First strand synthesis:

                      H1       H2      BJ x 2
   RNA               2.5ul     8ul     8ul
   10mM dNTP mix     1.0ul     1ul     1ul
   50uM Oligo(dT)20  1.0ul     1ul     1ul 
   H2O               5.5ul     -       -
   65C 5min -> place on ice for 1min
  
   Add
   10x RT buffer      2ul      2ul     2ul
   25mM MgCl2         4ul      4ul     4ul
   0.1M DTT           2ul      2ul     2ul
   RNase OUT(40U/ul)  1ul      1ul     1ul
   SS III RT(200U/ul) 1ul      1ul     1ul
   50C 50min -> 85c 5min  -> place on ice -> add 1ul RNase H, 37C 20min
  
   Purify with the Qiaquick column (combine the two BJ reactions), eluted with 30ul ddH2O. 

Second strand synthesis:

   DNA                     28ul
   10X NEBuffer             4ul
   50ng/ul random hexamers  2ul
   10mM dNTP              0.4ul
   H2O                    3.4ul

   65C 5min -> place on ice for 1min -> add 2ul 10U/ul DNA Pol I -> 16C 2h -> 75C 20min
   Purify with Qiaquick column.

Exp.2 PAGE purification of CES22k probes made in the past two weeks.[edit]

I decided to purify 60ul of #2, #3, #4, #5 in four gels.

  File:2007-11-19-CES22k-PAGE.png
  File:2007-11-19-CES22k-PAGE-purified-probes.png
  Concentration:
  CES22k #2:    2ng/ul ->  77nM
  CES22k #3:  5.7ng/ul -> 220nM
  CES22k #4:  9.3ng/ul -> 358nM
  CES22k #5:  7.5ng/ul -> 288nM
  Mix 30ul #3 + 18ul #4 + 22.5ul #5 => 277nM of pooled probes.