BrdU pull-down of 2 µg of gDNA was performed as described previously with two major differences (Dilley et al. 2016 (link); Verma et al. 2018 (link)). First, sonication was omitted in order to preserve C-circle structure. Instead, gDNA was digested using AluI and MboI (New England Biolabs). Second, the digested gDNA was not denatured prior to pull-down. After BrdU pull-down, the nascent DNA was cleaned with ChIP DNA Clean and Concentrator kit (Zymo) and eluted in 20 µL. For C-circle PCR, 6 µL of eluted pull-down was used per reaction. Digested gDNA input (30 ng per reaction) was run in parallel.
Protocol detail
BrdU Pull-down for C-circle Analysis
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Brdu
Dna chip
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Variable analysis
independent variables
- Omission of sonication to preserve C-circle structure
- Digestion of gDNA using AluI and MboI instead of sonication
- No denaturation of digested gDNA prior to pull-down
- Yield of nascent DNA after BrdU pull-down
- C-circle PCR results
- Amount of gDNA used for BrdU pull-down (2 µg)
- Volume of eluted pull-down used for C-circle PCR (6 µL)
- Amount of digested gDNA input used for C-circle PCR (30 ng)
- Not explicitly mentioned
- Not explicitly mentioned
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