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==== 1st PCR at 17 cycles, 5ul PCR product ==== [[File:ZhangLab_2 2014-12-22 14hr 53min_1stPCR-gel1.jpg| 450px]]<br> <br> [[File:ZhangLab_2 2014-12-22 14hr 53min_1stPCR-gel2.jpg| 450px]]<br> <br> [[File:ZhangLab_2 2014-12-22 14hr 53min_1stPCR-gel3.jpg| 450px]]<br> * All samples in 3 images above (W_1 to W_16 and T_1 to T_16) had 60fg of unmeth lambda DNA spike in [[File:ZhangLab_2 2014-12-22 14hr 53min_1stPCR-gel4.jpg|350px]]<br> <br> [[File:ZhangLab_2 2014-12-22 14hr 53min_1stPCR-gel5.jpg| 320px]] * NTC_1 and NTC_2 are NTC during cell lysis step. * Idid not see any different between 1nu reaction (W_XX and T_XX) and NTC in cell lysis, MspI and ligation control. Most of the signals came from primers, adaptors and adaptor dimers and faint band below 200bp band. * However, for reaction with 10 and 100nuclei (T10n and T100n) I noticed a faint smear below and above 200bp as cropped in image. In addition, the intensity of smear was proportional to number of nuclei in the reaction. I was hoping that it is the real signal of RRBS libraries from mouse nuclei. * The next challenging for single nuclei reaction was how to remove the background of adaptor dimers ~125bp which was a dominant background and the size close to the size of expected libraries and increase amplification of the real signal which was not visible in TBE gel. * I continued to purify the 1st round amplicons with AMPure beads twice (1:1 ratio). Left over PCR product volume was 45 ul.
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