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.