Noi/NOTES/2014-10-31

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Repeating of BIS/DNA SMART ChIP-Seq protocol

Back to calendar

  • According to experiment on 2014-10-29, I still see amplification of contaminants in BIS-NTC. In that experiment, I used BisulFlash DNA Methylation kit (bead-base) from Epigentek. For this experiment, I would ignore a bisulfite conversion rate and recovery yield of this kit. I just wanted to try the new kit with the price is affordable to see whether I can remove contamination. Even the contamination was observed, the level was lower than Zymo DNA Methylation-Lightning kit (column-based).
  • In this experiment, I want to repeat experiment again using Zymo DNA Methylation-Lightning Magprep kit. I want to see if the level of contamination is comparable to the first experiment that I tried with DNA Methylation-Lightning column-based kit in the first experiment on 2014-10-08. This might explain about contamination in CT conversion reagent and the procedures between column and bead.
  • I also wanted to compare the yield after amplification between BIS-100ng input and BIS-NTC.
  • I used the old "Zymo DNA Methylation-Lightning Magprep kit" I purchased in September 2013 as getting the new kit is too expensive for trial experiment. However, I believe that the reagents should be clean. I UV all reagent except conversion reagent.
  • I used ClonTech control fragmented human gDNA for this experiment. I followed the procedures of bisulfite conversion by zymo DNA Methylation-Lightning Magprep kit and resuspend with 22ul Elution buffer. Then transfer 20ul of converted DNA for library preparation
  • I used the same procedure of library preparation using ClonTech DNA SMRT-Seq kit as describe in [2014-10-29]