Kun:LabNotes/CpgSeq/2007-12-7
Exp.l PCR on the captured CpG islands[edit]
- Template: two capturing reactions started at 12/05/2007.
- Primer: Amp_F_sol, Amp_R_sol
x 1 x 2 template: 5ul 10ul ABI 10x buffer: 4.5ul 9ul 25mM MgCl2: 3ul 6ul 10mM dNTP: 1ul 2ul 100uM AmpFSol: 0.15ul 0.3ul 100uM AmpRSol: 0.15ul 0.3ul 50X SYBG I: 0.4ul 0.8ul iTaq: 1ul 2ul H2O: 35.3ul 70.6ul
94C 3min -> 10 cycles of (94C 45sec -> 59C 2min -> 72C 1min) -> 20 cycles of (94C 45 sec -> 72C 1min) -> 72C 3min -> 15C hold.
No amplification in both reactions, even after I added another 15 cycles. I then realized that I use the wrong primer AmpF2Sol. so I repeated the experiment using AmpFSol.
The second amplification didn't work either, even after 50 cycles.
Exp.2 Repeat the capturing experiment[edit]
- I decided to repeat phosphorylation reaction on the probes by adding 0.5ul 10mM ATP and 1ul T4PNK to the reactions (S1, S2, L), 37C 15min, 85C 10min.
- I also decided to do one round of DNA synthesis on the bisulfite converted DNAs to convert them into dsDNA prior to the capturing reactions.
DNA 20ul 10x NEBbuffer2 3ul 10mM dNTP 1.5ul 100uM N6 3ul
94C 3min -> snap on ice for >1min -> add 2ul 10U/ul DNA Pol I -> 16C 10min -> 37C 10min -> 75C 10min. Purify with MinElute columns, eluted with 22ul ddH2O. Measure concentration.
CpGMIPv2 S2(211nM) 3.3ul CpGMIPv2 L1(243nM) 1.4ul H2O 95.3ul
RL DNA(31ng/ul) Jurkat DNA(32ng/ul) Template 10ul 10ul 10x buffer 1.5ul 1.5ul 10nM CpG-MIP 2ul 2ul (set up another 2 with the v1 probes) H2O 1.5ul 1.5ul
94C 3min->65C 20h-> add 1ul SLN mix (2U/ul stoffel, 2U/ul AmpLigase, 2mM dNTP) -> 65C 30min -> 15 cycles of (94C 1min -> 65C 1h) -> 65C 4h -> 37C 1min -> add 1ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 94C 5min -> 4C hold.