Kun:LabNotes/CpgSeq/2007-12-11
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Exp.1 Repeat the PCR experiment on the circularized reactions using Hd48MIP1,2,5 primers with a touchdown protocol[edit]
Template: 1ul (1:100 dilution of bifulfite converted gDNAs) 10X buffer: 2.5ul 10mM dNTP: 0.5ul 10uM Primer pair 0.75ul 50X SYBR I 0.2ul AmpliTaq Gold 0.25ul H2O 20ul
Total number of reactions: 4 MIP reactions x 3 primer pairs + 2 control x 3 primer pairs = 18 95C 15min -> 8 cycles of (94C 30sec -> 60C 30sec (-0.5C per cycle) -> 72C 30 sec) -> 27 cycles of (94C 30sec -> 56C 30sec -> 72C 30 sec) -> 72C 3min -> 15C hold.
File:2007-12-11-CpG-MIP-PCR-specific-primer-touchdown.png
Exp.2 Perform another round of capturing reaction at 60C[edit]
- dsDNA synthesis:
DNA (11/12/07) 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 15min -> 37C 30min -> 75C 10min. Purify with MinElute columns, eluted with 22ul ddH2O. Measure concentration.
RL dsDNA(47ng/ul) Jurkat dsDNA(95ng/ul) Template 10ul 5ul 10x buffer 1.5ul 1.5ul 10nM CpG-MIP 2ul 2ul (set up another 2 with the v1 probes) H2O 1.5ul 6.5ul
RL Bis-DNA Jurkat Bis-DNA Template 5ul 5ul 10x buffer 1.5ul 1.5ul 10nM CpG-MIPv2 2ul 2ul H2O 6.5ul 6.5ul
Jurkat gDNA Jurkat gDNA (100ng/ul) Template 5ul 5ul 10x buffer 1.5ul 1.5ul 10nM CpG-MIPv1 2ul CpG-MIPv1 2ul H2O 6.5ul 6.5ul
95C 3min->60C 42h-> add 1ul SLN mix (2U/ul stoffel, 2U/ul AmpLigase, 1mM dNTP) -> 65C 1h -> 40 cycles of (95C 1min -> 65C 15min) -> 65C 4h -> 37C 1min -> add 1ul of exo mix (Exo I, Exo III, T7 exo), 0.5ul USER enzyme->37c 1 hour -> 94C 5min -> 4C hold.