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| H2O 1.5ul 1.5ul | | H2O 1.5ul 1.5ul |
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| 94C 3min->65C 20h-> add 1ul SLN mix (1U/ul stoffel, 0.5U/ul AmpLigase, 1uM dNTP) -> 60C 4h -> 15 cycles of (94C 1min -> 60C 1h) -> 60C 4h -> 37C 1min -> add 1ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 94C 5min -> 4C hold. | | 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. |
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| ==Exp.3 Measure the solexa libraries purified today with PicoGreen on Chromo4==
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| *Make 500ul of 1:200 diluted PicoGreen: 2.5ul PicoGreen + 497.5ul 1X TE.
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| *Make serial dilution of DNA standard:
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| 1X TE 2ng/ul DNA Diluted PicoGreen Total volume
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| 0ul 12.5ul 12.5ul 25ul
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| 10ul 2.5ul 12.5ul 25ul
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| 11.5ul 1ul 12.5ul 25ul
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| 12ul 0.5ul 12.5ul 25ul
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| 1X TE 2ng/ul ssDNA Diluted PicoGreen Total volume
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| 0ul 12.5ul 12.5ul 25ul
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| 10ul 2.5ul 12.5ul 25ul
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| 11.5ul 1ul 12.5ul 25ul
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| 12ul 0.5ul 12.5ul 25ul
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| 1X TE sample Diluted PicoGreen Total volume
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| 10ul 2.5ul 12.5ul 25ul
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| Read twice at SYBG I channel.
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| Sample layout:
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| {| {{table}}
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| | align="center" style="background:#f0f0f0;"|'''Ds-ref'''
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| | align="center" style="background:#f0f0f0;"|'''Ss-ref'''
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| | align="center" style="background:#f0f0f0;"|'''Samples'''
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| | align="center" style="background:#f0f0f0;"|'''Samples'''
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| | align="center" style="background:#f0f0f0;"|'''Samples(11/21)'''
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| |-
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| | 1ng/ul||1ng/ul||H1c(12/03)||Bjg(12/07)||5
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| |-
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| | 0.2ng/ul||0.2ng/ul||H2c(12/03)||Bjc(12/07)||6
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| |-
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| | 0.08ng/ul||0.08ng/ul||Hues6(12/03)||Bjg(11/29)||7
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| |-
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| | 0.04ng/ul||0.04ng/ul||H1c(12/07)||Bjc(11/29)||8
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| |-
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| | 1ng/ul||1ng/ul||H2g(12/07)||CES22k#2||9
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| |-
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| | 0.2ng/ul||0.2ng/ul||H2c(12/07)||CES22k#4||10
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| |-
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| | 0.08ng/ul||0.08ng/ul||Hues6g(12/07)||CES22k#5||11
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| |-
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| | 0.04ng/ul||0.04ng/ul||Huestc(12/07)||||12
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| |-
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| |}
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| {| {{table}}
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| | align="center" style="background:#f0f0f0;"|''''''
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| | align="center" style="background:#f0f0f0;"|'''Ds-ref'''
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| | align="center" style="background:#f0f0f0;"|'''Ss-ref'''
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| | align="center" style="background:#f0f0f0;"|'''Samples'''
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| | align="center" style="background:#f0f0f0;"|'''Samples'''
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| | align="center" style="background:#f0f0f0;"|'''Samples'''
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| |-
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| | A||1.48||0.23||0.13||0.45||0.17
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| |-
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| | B||0.42||0.14||0.13||0.13||0.35
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| |-
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| | C||0.24||0.12||0.19||0.81||0.44
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| |-
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| | D||0.17||0.13||0.13||0.25||0.65
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| |-
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| | E||1.62||0.21||0.27||0.13||0.52
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| |-
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| | F||0.41||0.13||0.14||0.14||0.35
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| |-
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| | G||0.24||0.13||0.25||0.14||0.15
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| |-
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| | H||0.16||0.12||0.15||0.09||0.35
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| |-
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| |}
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| Calculated DNA concentration:
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| {| {{table}}
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| | align="center" style="background:#f0f0f0;"|'''Samples'''
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| | align="center" style="background:#f0f0f0;"|'''ng/ul'''
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| | align="center" style="background:#f0f0f0;"|'''nM'''
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| | align="center" style="background:#f0f0f0;"|'''Gel quant (ng/ul)'''
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| |-
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| | H1c(12/03)||0.13||1.49||0.18
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| |-
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| | H2c(12/03)||0.1||1.21||0.13
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| |-
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| | Hues6(12/03)||0.53||6.17||0.82
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| |-
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| | H1c(12/07)||0.12||1.44||0.11
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| |-
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| | H2g(12/07)||1.08||12.51||0.98
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| |-
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| | H2c(12/07)||0.13||1.52||0.13
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| |-
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| | Hues6g(12/07)||0.9||10.51||
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| |-
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| | Hues6c(12/07)||0.25||2.85||
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| |-
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| | Bjg(12/07)||2.3||26.78||
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| |-
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| | Bjc(12/07)||0.09||1.1||
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| |-
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| | Bjg(11/29)||4.82||56.07||
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| |-
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| | Bjc(11/29)||0.93||10.81||
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| |-
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| | Hues6 gDNA(11/20)||0.35||4.03||1.2
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| |-
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| | Hues6 cDNA(11/20)||1.6||18.59||2.8
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| |-
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| | H1 gDNA(222ng/ul)||2.27||26.41||4.5
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| |-
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| | H1 cDNA(11/20)||3.69||42.91||4.6
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| |-
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| | H2 gDNA(200ng/ul)||2.84||32.99||3.7
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| |-
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| | H2 cDNA(11/20)||1.65||19.15||3.2
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| |-
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| | BJ MDA DNA(11/20) ||0.27||3.11||0.34
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| |-
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| | BJ cDNA(11/20l)||1.59||18.55||3
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| |-
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| |}
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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.