Kun:LabNotes/ASE/2007-11-26
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]
- Make 500ul of 1:200 diluted PicoGreen: 2.5ul PicoGreen + 497.5ul 1X TE.
- Make 100ul 2ng/ul lambda DNA solution: 2ul 100ng/ul stock DNA + 98ul 1X TE.
- 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.