Kun:LabNotes/CpgSeq/2007-12-7

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Exp.l PCR on the captured CpG islands

  • 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

  1. 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.
  2. 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 (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.

Exp.3 Measure the solexa libraries purified today with PicoGreen on Chromo4

  • Make 500ul of 1:200 diluted PicoGreen: 2.5ul PicoGreen + 497.5ul 1X TE.
  • Make serial dilution of DNA standard:
     1X TE        2ng/ul DNA       Diluted PicoGreen       Total volume
      0ul           12.5ul               12.5ul                   25ul 
      10ul           2.5ul               12.5ul                   25ul 
    11.5ul             1ul               12.5ul                   25ul 
      12ul           0.5ul               12.5ul                   25ul 
     1X TE        2ng/ul ssDNA       Diluted PicoGreen       Total volume
      0ul           12.5ul               12.5ul                   25ul 
      10ul           2.5ul               12.5ul                   25ul 
    11.5ul             1ul               12.5ul                   25ul 
      12ul           0.5ul               12.5ul                   25ul 
    1X TE            sample          Diluted PicoGreen       Total volume
      10ul           2.5ul               12.5ul                   25ul 

Read twice at SYBG I channel.

Sample layout:

Ds-ref Ss-ref Samples Samples Samples(11/21)
1ng/ul 1ng/ul H1c(12/03) Bjg(12/07) 5
0.2ng/ul 0.2ng/ul H2c(12/03) Bjc(12/07) 6
0.08ng/ul 0.08ng/ul Hues6(12/03) Bjg(11/29) 7
0.04ng/ul 0.04ng/ul H1c(12/07) Bjc(11/29) 8
1ng/ul 1ng/ul H2g(12/07) CES22k#2 9
0.2ng/ul 0.2ng/ul H2c(12/07) CES22k#4 10
0.08ng/ul 0.08ng/ul Hues6g(12/07) CES22k#5 11
0.04ng/ul 0.04ng/ul Huestc(12/07) 12
' Ds-ref Ss-ref Samples Samples Samples
A 1.48 0.23 0.13 0.45 0.17
B 0.42 0.14 0.13 0.13 0.35
C 0.24 0.12 0.19 0.81 0.44
D 0.17 0.13 0.13 0.25 0.65
E 1.62 0.21 0.27 0.13 0.52
F 0.41 0.13 0.14 0.14 0.35
G 0.24 0.13 0.25 0.14 0.15
H 0.16 0.12 0.15 0.09 0.35

Calculated DNA concentration:

Samples ng/ul nM
H1c(12/03) 0.13 1.49E-09
H2c(12/03) 0.1 1.21E-09
Hues6(12/03) 0.53 6.17E-09
H1c(12/07) 0.12 1.44E-09
H2g(12/07) 1.08 1.25E-08
H2c(12/07) 0.13 1.52E-09
Hues6g(12/07) 0.9 1.05E-08
Huestc(12/07) 0.25 2.85E-09
Bjg(12/07) 2.3 2.68E-08
Bjc(12/07) 0.09 1.10E-09
Bjg(11/29) 4.82 5.61E-08
Bjc(11/29) 0.93 1.08E-08
Hues6 gDNA(11/20) 0.35 4.03E-09
Hues6 cDNA(11/20) 1.6 1.86E-08
H1 gDNA(222ng/ul) 2.27 2.64E-08
H1 cDNA(11/20) 3.69 4.29E-08
H2 gDNA(200ng/ul) 2.84 3.30E-08
H2 cDNA(11/20) 1.65 1.92E-08
BJ MDA DNA(11/20) 0.27 3.11E-09
BJ cDNA(11/20l) 1.59 1.85E-08