Noi/NOTES/2014-7-10
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CRC oligo pool from Illumina[edit]
- I received one tube of CRC (colorectal cancer) oligo pool from Xuyu. The total volume of oligo ~1mL
- The concentration of oligo mix is 100uM and there are 1,024 oligos in this pool.
- I verify concentration by Qubit ssDNA assay by measuring non-diluted and 10X, 50X and 100X diluted oligo in TE Buffer.
Sample Conc. in the Qubit Unit uL used Dilution Sample Conc. (ng/ul) Conc. of original tube (ng/ul) 1ul non-diluted Out Of Range 1 2ul non-diluted Out Of Range 2 2ul 10X dilution Out Of Range 2 2ul 100X dilution 140 ng/mL 2 100 14 1,400 2ul 50X dilution 270 ng/mL 2 100 27 1,350
- I also verify the integrity of oligos in 6% TBU gel to see if the level of background is high.
File:ZhangLab 2 2014-07-10 17hr 22min CRC-oligos.jpg 1 = 1ul of non-diluted CRC oligos 2 = 2ul of non-diluted CRC oligos 3 = 2ul of 10X diluted CRC oligos 4 = 2ul of 100X diluted CRC oligos 5 = 2ul of 50X diluted CRC oligos
- It looked like there is very high background of truncated oligos as expect. I may need to do size selection of this oligo pool (~12ug) before capture to avoid any background during capture. In addition, I have a lot of oligos (at least 1,000ug) for this experiment.
PAGE size selection of CRC oligos[edit]
- I mix 3ug of CRC oligo in total 100ul of TBU loading dye
- For 4 gel I mixed 8.57ul of 1.4ug/ul CRC oligo with 192ul H2O and 200ul 2X TBU loading dye and split 100ul in 200ul PCR tube.
File:ZhangLab 2 2014-07-11 12hr 02min CRC PAGE-SS 1.jpg File:ZhangLab 2 2014-07-11 12hr 02min CRC PAGE-SS 2.jpg File:ZhangLab 2 2014-07-11 12hr 02min CRC PAGE-SS 3.jpg File:ZhangLab 2 2014-07-11 12hr 02min CRC PAGE-SS 4.jpg
- - Precipitate DNA in 12 LoBind tubes
- - Resuspend each tube with 20ul --> total 240ul
- - Quantify concentration with Qubit ssDNA assay --> conc. = XX ng/ul
- - Verify purified oligos in 6% TBU gel