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DNA yield of ChIP assay so low


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#1 thinh

thinh

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Posted 24 June 2014 - 12:13 AM

Hi guys

I’m doing ChIP assay with transcription factor. It’s new to me, and so many questions to me now.

Actually, my protocol seem to work well, but the DNA yield is low in my thought, and alsway feel not confident with my samples. So I’d like to describe here my experiment with hope to get all guys’ comments.

  1. 10ml media with seeded bacteria was subculted, induce the expression of my protein by L-arabinose within 6hours (my protein was constructed in pBAD plas)
  2. Add formaldehyde 1%, in 20min for crosslinking, and stop with Glycine 0.5M.
  3. Harvest cell, wash with TBS, suspend in 1ml lysis buffer containing Protease inhibitor and lysozyme, 37oC in 30min incubation.
  4. Sonication with Vibra Cell sonicator 30%, 10s on/10 off, 5min.
  5. 14000rpm, 15min centrifugation and do IP reaction: 100ug chromatin + 10ug of antibody, Overnight incubation with rotator in 4oC.
  6. Add Protein A/G agarose bead (Santa Cruiz) 20ul, Overnight incubation with rotator.
  7. Harvest bead, wash with RIPA buffer and TE buffer, elute with 100ul of elution buffer containing 1% SDS at 65oC in 1hours.

Centrifuge, take the supernatant and incubate at 65oC, overnight for de-crosslinking.

  1. Treat with RNAse and Protease K and then purification.

I tried two method:

- PCR purification kit (Qiagen): elution sample checked by nanodrop did not showed any DNA concentration.

- DNA precipitation with ethanol (+ sodium acetate and glycogen) and then suspend the obtained DNA in 10ul of water: these sample showed DNA concentration of about 10ng/ul by nanodrop, but 260/280 and 260/230 very low (1.0 and 0.1, respectively).

After that for later step of sequencing, the company required me to check DNA conc by Qubit kit, and but results by Qubit showed much lower compared to nanodrop (just around 0.2ng/ul).

I don’t care about this concentration because I still have enough DNA sample to send for sequencing company (10ng), even follow Qubit result. But I’m worry about my sample quality, as you know the contamination is so much, and I don’t know how to check sample quality.

Actually, I did two methods to control my process. First is western blot with the bead after washing step, it showed my expected target band. Second, I did PCR with primers to detect one sequence binding to my target protein that was published in one paper, but the result is not stable, sometime I got the band, sometime no band, and sometime the band appeared in even my control sample (sample not added with antibody).

Please tell me what problem in my process. The DNA concentration I got is normal or not. And which method I should use to check my DNA sample for later step of Illumina sequencing.

Hope to receive much options for my process.

Thanks so  much.






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