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Functional Genomics for Drosophila
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Quality control of the fixed chromatin from Drosophila embryos

Overview

Before progressing to the chromatin immunopurification, it is important to quality control the chromatin itself. This will prevent valuable resources being wasted on sub-standard material, e.g., the extraction failed or, the chromatin is of the wrong size. The procedures outlined below can be used to assay chromatin batches.

Protocol - reversal of chromatin-protein crosslinks

To assay the quality and quantity of genomic DNA that has been purified, it is necessary to reverse the crosslinking, remove the protein with a proteinase K treatment and RNA with RNase.

  1. To 50 µl chromatin, add 2 µl RNase A and 150 µl nuclear lysis buffer (50 mM Tris.HCL, pH 8.1; 10 mM EDTA.Na2; 1% SDS). Incubate at 67 °C for 4-5 hours (or overnight), to reverse the crosslinking. Store at -20 °C overnight.
  2. Thaw out the samples and add 4.2 µl proteinase K (Roche Diagnostics: recombinant PCR grade, cat. no. 3115887, 50 U/ml) and 0.8 µl 10% SDS. Incubate at 45 °C for 2 hours.
  3. Purify using Qiagen columns (QIAquick PCR purification kit) and elute in 50 µl elution buffer supplied with the columns or water. Store at -20 °C.

Protocol - gel electrophoresis

After reversal of the chromatin-protein crosslinks genomic DNA from D. melanogaster will be visible as a smear between 200 bp and 2 kb, most will be between 500 bp and 1 kb.

  1. Make a 1% agarose gel and add 5 µl ethidium bromide (10 mg / ml) per 100 ml of gel.
  2. To 5 µl sample, add 1 µl 6 x loading buffer. Load.
  3. Run the gel at 80 V until the fastest dye has moved 2/3 of the gel length.
  4. Visualise the gel using a UV transilluminator and photograph.

Protocol - optical density measurements

Optical density measurements are made using a Nanodrop ND-1000 spectrophotometer (http://www.nanodrop.com) that has been calibrated using dilutions of genomic DNA. This calibration showed that the linear range is 10 to 1000 ng/µl. The calibration curve is used to interpret the measurement using an in-house script.

Good quality DNA will have an OD 260/280 ratio of 1.8 to 2 and an OD 260/230 of 1.8 or greater. This is because nucleic acid is detected at 260 nm, whereas protein, salt and solvents are detected at 230 and 280 nm.

  1. Open the Nanodrop icon and select 'Nucleic Acid Measurements'.
  2. Add 2 µl of solvent the sample has been dissolved in ("solvent"); the instrument will then initialise.
  3. After each and all subsequent measurements clean the pedestal by wiping with a dry lint-free tissue.
  4. Add 2 µl of solvent and press 'Blank'.
  5. Repeat the blanking until there is a stable baseline close to zero.
  6. Confirm that the baseline is correct by measuring 2 µl of the solvent, as if it were your first sample by pressing 'Measure'.
  7. Add 2 µl of the first sample making sure to add the sample ID (or name) to the 'Sample ID' field and then press 'Measure'.
  8. Repeat step 3 and then 7 for all samples.
  9. Confirm that the baseline is correct after taking all measurements by measuring 2 µl of the solvent, as if it were your last sample by pressing 'Measure'.
  10. Each of the measurements is automatically saved by the instrument and these can then be calibrated using the in-house script.

Version 1.1. R. Auburn. (25-08-2006)