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==Exp.1 PAGE purification and quantification of CES22k solexa libraries== 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. [[Image:2007-11-24-CES22k-exonic-capture-PAGE-purification.png|600px]] [[Image:2007-11-26-CES22k-sollib-quant.png|600px]] [[Image:test-today.png|200px]] 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== #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. {| {{table}} | align="center" style="background:#f0f0f0;"|''' ''' | align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''1.1''' | align="center" style="background:#f0f0f0;"|'''1.2''' | align="center" style="background:#f0f0f0;"|'''2.1''' | align="center" style="background:#f0f0f0;"|'''2.2''' | align="center" style="background:#f0f0f0;"|'''3.1''' | align="center" style="background:#f0f0f0;"|'''3.2''' | align="center" style="background:#f0f0f0;"|'''4.2''' | align="center" style="background:#f0f0f0;"|'''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 [[Image:2007-11-26-picotest-xyplot.png|400px]] <b>Conclusion:</b> 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. {| {{table}} | align="center" style="background:#f0f0f0;"|'''Library''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''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 |- | |} <b>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.</b> ==Exp.3 Repeat the CES22k capturing experiment on BJ Fibroblast gDNA/cDNA== *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. {| border="1" cellpadding="5" cellspacing="0" align="center" |- | align="center" style="background:#f0f0f0;"|''' ''' | align="center" style="background:#f0f0f0;"|''' ''' | align="center" style="background:#f0f0f0;"|'''Template''' | align="center" style="background:#f0f0f0;"|'''10x buffer''' | align="center" style="background:#f0f0f0;"|'''CES22k probes (319nM)''' | align="center" style="background:#f0f0f0;"|'''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.
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