Kun:LabNotes/CpgSeq/2007-11-9
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Exp. 1 Perform PCR on the bisulfite converted DNAs (positive control)[edit]
- Template: Jurkat DNA, DNA RL, concentration unknown.
- Primer: hd48MIP-1, hd48MIP-2, hd48MIP-5.
x1 x6 template: 1ul each 10x ABI buffer: 2.5ul 15ul 25mM MgCl2: 1.5ul 9ul 10mM dNTP: 0.5ul 3ul 10uM Primer mix: 0.5ul 1ulx3 50X SYBG I: 0.2ul 1.2ul iTaq: 0.2ul 1.2ul H2O: 18.6ul 114ul
94C 3min -> 40 cycles of (94C 30sec -> 57C 30min -> 72C 1min) -> 72C 3min -> 15C hold.
Exp. 2 Measure the concentration of PAGE purified padlock probes by gel electrophoresis[edit]
Set 1: 3.7ng/ul = 128nM Set 2: 4.2ng/ul = 162nM
Mix two sets in 2:1 molar ratio, and dilute to 1nM (total concentration):
13ul of set I + 5ul of Set 2 + 230ul H2O -> 10nM CpG-MIP mix 20ul of 10uM mix + 180ul H2O -> 1nM CpG-MIP mix
Exp. 3 CpG island capture[edit]
RL DNA Jurkat DNA (all converted, conc. unknown) Template 15ul 15ul 10x buffer 2ul 2ul 1nM CpG-MIP 1ul 1ul
94C 3min->60C 12h-> add 2.5ul/1.5ul SLN mix (1U/ul stoffel, 0.5U/ul AmpLigase, 100nM dNTP) -> 60C 4h -> 15 cycles of (94C 1min -> 60C 1h) -> 60C 4h -> 37C 1min -> add 3ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 94C 5min -> 4C hold.