Difference between revisions of "ChIP-Chip E. coli"
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Revision as of 05:33, 29 May 2008
This protocol is in developement and currently under investigation in the lab. Please contibute and watch changes in the next weeks!
Torsten Waldminghaus 06:29, 17 March 2008 (CDT)
Anyone should feel free to add themselves as a curator for this consensus protocol. You do not need to be a curator in order to contribute. The OpenWetWare community is currently discussing the idea of protocol curators. Please contribute.
ChIP-Chip stands for chromatine immuno precipitation and Chip in the sence of DNA-array. It is a technique to determin the DNA binding sites of a DNA binding protein on a global scale. While the basic priciple is the same there are some differences in handling cells from various organisms. This protocol is developed and tested for E. coli. It should work the same way for other bacteria but that remains to be proven (please add any experience). The main difference to other protocols is that no amplification is done after reversion of cross-linking. The basic procedure is described in .
Other ChIP-Chip protocols can be found here:
- 2 ml Ultralink protein A/G beads (catalog number 53132, Pierce)
- Specific antibody for example RNA polymerase β subunit from Neoclone, Madison
Material for about 50 experiments:
- 75 ml Formaldehyd (37%)
- 500 ml 2.5 M Glycine
- 1.5 l TBS (400 ml 5xTBS)
- 50 ml TE
- 20 mg/ml Proteinase K in TBS (store up to 1 year at -20 C)
- 1 ml 100mM PMSF or 0.5 ml 250 mM Pefabloc
- 50 ml Lysis Buffer:
- 10mM Tris (pH 8.0)
- 20% sucrose
- 50mM NaCl
- 10mM EDTA
- 10 mg/ml lysozyme
- 500 ml IP-Buffer (Immunoprecipitation Buffer):
- 50 mM HEPES-KOH pH 7.5
- 150 mM NaCl
- 1 mM EDTA
- 1% Triton X 100
- 0.1 % Sodium deoxycholate
- 0.1 % SDS
- 30 ml IP-Buffer + 500 mM NaCl
- 30 ml Wash-Buffer:
- 10mM Tris pH 8.0
- 250 mM LiCl
- 1 mM EDTA
- 0.5% Nonidet-P40
- 0.5% Sodium deoxycholate
- 10 ml Elution-Buffer:
- 50 mM Tris (pH 7.5)
- 10 mM EDTA
- 1% SDS
Any equipment used to perform the protocol (link to a method for using them).
Formaldehyde cross link and sonication:
- Add 27μl formaldehyde (37%) per ml medium (substract what you took out for messuring OD) => final concentration of about 1%
- Shake slowly (100 RPM) for 20 min at RT
- Add 10 ml of 2.5 M glycine => final concentration of about 0.5 M
- Keep shaking for 5 min
- Harvest 10 ml of cells for each DNA-preparation (centrifuge 2500 g, 4°C, 10 min)
- Wash twice in cold 10 ml TBS (20mM; see Material) pH7.5
- Resuspend in 1 ml Lysis-Buffer
- Incubate at 37 °C for 30 min (not shaking)
- Add 4 ml of IP-Buffer
- Add 50 μl of 100mM PMSF => final concentration of 1mM
Note: for sonication everybody, every sonicator, every day and everything else is different so see critical steps (below) and than try what work best for you.
- Centrifuge 12.000 g, 4°C, 10 min
- Use 800 μl aliquot as input sample for one immunoprecipitation experiment
- Add 20 μl of Ultralink protein A/G beads
- Add specific antibody (for example 5 μl of RNA polymerase β subunit; see Material above)
- Incubate at 4 °C over night on a slow rotator
- Collect each sample on Spin-x column by centrifugation for 2 min at 3500 rpm
- All following wash steps should be on a rotator at room temperature for 3min with 2 min centrifugation as above:
- Wash twice with 500 μl I-Buffer
- Wash with 500 μl I-Buffer with 500 mM NaCl
- Wash with 500 μl Wash-Buffer
- Wash with 500 μl TE
- Place filter unit containing the beads into a new 1.5-ml microcentrifuge tube and add 100 μl of elution buffer. Gently pipet up and down two or three times in order to dislodge beads from the filter. Incubate 10 min in a 65 °C water bath. A water bath is used instead of other heating apparatuses in order to improve heat transfer. Make sure the sample in the tube is actually under the water and not in the air above.
- Centrifuge beads 2 min at 3500 rpm, room temperature. Discard filter with
beads. Transfer the eluate into new 1.5 ml tube and leave on ice until you start the next step short before you go home for dinner.
- Add 80 μl TE and 20 μl Proteinase K to the tube and divide on PCR tubes fitting your PCR machine (for example 2x100μl).
- To reverse cross-links, place tubes into PCR machine. Incubate 2 hr at 42 °C, followed by 6 hr at 65 °C. Store samples at 4 °C until use.
- Purify DNA by phenol extraction and ethanol precipitation
- Dilute in 20 μl A. dest
- Measure DNA-content idealy at a NanoDrop (should be around 0.2 to 0.4 μg)
- Use 1 and 10 ng DNA as template for quantitative PCR with primers that are specific for a known binding site of your DNA-binding protein and one negative control.
Referenced from the main protocol, a more thorough explanation of particularly important steps in the protocol.
Referenced from the main protocol, an explanation of what can cause things to go wrong with the protocol.
Referenced from the main protocol, any comments about the protocol should be made here; i.e. how it was developed. Any comments added should be signed (by adding *'''~~~~''': in front) and explained. Links to FAQs/tips provided by other sources such as the manufacturer or other websites would be best made here.
Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.
It might also be good to add an image to show the workflow and timescales for experiment planning.
Acnkowledge any help you had in development, testing, writing this protocol.
- Grainger DC, Hurd D, Harrison M, Holdstock J, and Busby SJ. Studies of the distribution of Escherichia coli cAMP-receptor protein and RNA polymerase along the E. coli chromosome. Proc Natl Acad Sci U S A. 2005 Dec 6;102(49):17693-8. DOI:10.1073/pnas.0506687102 |
- Grainger DC, Hurd D, Goldberg MD, and Busby SJ. Association of nucleoid proteins with coding and non-coding segments of the Escherichia coli genome. Nucleic Acids Res. 2006;34(16):4642-52. DOI:10.1093/nar/gkl542 |
- Glycogen does not interfere with Klenow random labeling http://www.fermentas.com/catalog/reagents/glycogen.htm
Add links to all the OWW protocols that have been used in making the consensus.
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