Kun:LabNotes/ASE/2007-11-26

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Exp.1 PAGE purification and quantification of CES22k solexa libraries[edit]

I purified 12 PCR amplicons from the exonic SNP capturing experiment did on 11-20-2007. 40ul per amplicon into each lane of the 5-well gels.

 File:2007-11-24-CES22k-exonic-capture-PAGE-purification.png
 File:2007-11-26-CES22k-sollib-quant.png
 File:Test-today.png
 Quantification:
 1. NA20431   4.4ng/ul     110nM
 2. GM20431   4.2ng/ul     100nM
 3. GC1F      3.9ng/ul      94nM
 4. GC1EP     3.4ng/ul      80nM
 >------shipped to VCU on 11-26-2007-----<
 5. Hues6g    1.2ng/ul     28nM
 6. Hues6c    2.8ng/ul     68nM
 7. Hybrid 1g 4.5ng/ul    110nM
 8. Hybrid 1c 4.6ng/ul    110nM
 9. Hybrid 2g 3.7ng/ul     87nM
 10.Hybrid 2c 3.2ng/ul     75nM 
 11.BJg       0.34ng/ul     8nM
 12.BJc       3.0ng/ul     71nM

Exp.2 Test the protocol of quantifying DNA with PicoGreen on Chromo4[edit]

  1. Make 500ul of 1:200 diluted PicoGreen: 2.5ul PicoGreen + 497.5ul 1X TE.
  2. Make 100ul 2ng/ul lambda DNA solution: 2ul 100ng/ul stock DNA + 98ul 1X TE.
  3. Make serial dilution of DNA standard:
     1X TE        2ng/ul DNA       Diluted PicoGreen       Total volume
      0ul           25ul               25ul                   50ul 
      20ul           5ul               25ul                   50ul 
     22.5ul        2.5ul               25ul                   50ul
      24ul           1ul               25ul                   50ul

Get fluorescent reads on Chromo4 on row 1-4, two times per position.

Conc. (ng/ul) 1.1 1.2 2.1 2.2 3.1 3.2 4.2 4.1
A 1 3.43 3.45 3.62 3.63 3.72 3.69 3.37 3.41
B 0.2 0.52 0.52 0.51 0.51 0.53 0.53 0.51 0.52
C 0.1 0.34 0.33 0.32 0.32 0.33 0.32 0.32 0.33
D 0.04 0.2 0.2 0.19 0.19 0.2 0.2 0.22 0.22
E 1 2.05 2.06 2.06 2.06 2.17 2.16 2.14 2.14
F 0.2 0.51 0.51 0.52 0.52 0.52 0.52 0.56 0.53
G 0.1 0.33 0.33 0.32 0.32 0.34 0.33 0.33 0.33
H 0.04 0.2 0.2 0.2 0.2 0.19 0.2 0.2 0.2
  Scatter plot: X: concentration (ng/ul); Y: fluorescent intensity
  File:2007-11-26-picotest-xyplot.png

Conclusion: I think Chromo4 did get linear read-outs on PicoGreen stained DNA. This could be used to measure DNA concentration. However, a ssDNA standard should probably be used for ssDNA samples.


I measured the concentration of the eight Solexa libraries using this method. However the concentration seems to be lower than what I expected. Perhaps it is due to the fact that I exposed the picogreen to light (and probably UV) for a while.

Library ng/ul corrected,ng/ul
Hues6 gDNA 0.53 1.07
Hues6 cDNA 1.34 2.69
H1 gDNA 1.59 3.17
H1 cDNA 1.97 3.94
H2 gDNA 1.66 3.32
H2 cDNA 0.98 1.96
BJ MDA DNA 0.18 0.36
BJ cDNA 0.89 1.78

Note: I just realized that the concentrations should be multiplied by two, and then they are fairly close to what I got from the PAGE quantification.

Exp.3 Repeat the CES22k capturing experiment on BJ Fibroblast gDNA/cDNA[edit]

  • Mix 5ul CES22k #4 (358nM) with 6.22ul CES22k #5 (288nM) => 319nM.
  • Concentrate 120ul BJ fibroblast gDNA (6ng/ul)with YM-100 => Obtained 12ul of 40ug/ul gDNA.
Template 10x buffer CES22k probes (319nM) H2O
1 BJ conecentrated DNA(40ng/ul) 10ul 1.6ul 4.7ul 0ul
2 BJ cDNA(18ng/ul) 2.6ul 1.6ul 4.7ul 6.1ul

95C 10min->60C 18h-> add 0.5ul SLN mix (1U/ul stoffel, 0.5U/ul AmpLigase, 100nM dNTP) -> 60C 4h -> 15 cycles of (95C 2min -> 60C 1h) -> 60C 4h -> 37C 1min -> add 1.5ul of exo mix (Exo I, Exo III, T7 exo)->37c 1 hour -> 94C 5min -> 4C hold.