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==Abstract== | ==Abstract== | ||
ChIP-Chip stands for | ChIP-Chip stands for <u>Ch</u>romatin <u>I</u>mmuno<u>p</u>recipitation and chip in the sense of DNA microarray. It is a technique to determine the genome-wide binding sites of a DNA-binding protein. While the basic principle is the same for all species, there are some differences in handling cells. This protocol is developed and tested for '''E. coli'''. It should work the same way for other bacteria but that remains to be proven. Published protocols also exist for other bacterial species, including <i>Bacillus subtilis</i>, <i>Caulobacter crescentus</i> and <i>Mycobacterium tuberculosis</i>. | ||
Because <i>E. coli</i> can be grown to high cell densities relative to eukaryotes, it is possible to generate sufficient DNA to label without using a PCR-based method <cite>Grainger-2005</cite>. This method uses strand displacement primer extension with Klenow DNA polymerase and amplifies the DNA ~10-fold, while simultaneously incorporating dye-coupled nucleotide. | |||
Other ChIP-Chip protocols can be found here: | Other ChIP-Chip protocols can be found here: | ||
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Material for about 50 experiments: | Material for about 50 experiments: | ||
*75 ml | *75 ml Formaldehyde (37%) | ||
*500 ml 2.5 M [[Glycine]] | *500 ml 2.5 M [[Glycine]] | ||
*1.5 l [[TBS]] (400 ml 5xTBS) | *1.5 l [[TBS]] (400 ml 5xTBS) | ||
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==Procedure== | ==Procedure== | ||
This protocol has been broken up into 3 "days" but for <i>E. coli</i> it is possible to perform the entire ChIP experiment in a single day as well as the amplification/labeling step (which can be done overnight). | |||
'''1. Day''' | '''1. Day''' | ||
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*Add 50 μl of 100mM [[PMSF]] => final concentration of 1mM | *Add 50 μl of 100mM [[PMSF]] => final concentration of 1mM | ||
[[Image:Critical step.png]] Sonicate | [[Image:Critical step.png]] Sonicate so that DNA fragments are <1 kb on average (see "critical steps" section below). Using a Branson 450 sonifier with a microtip probe (inserted in the sample) 2 cycles of 30 s sonication are sufficient. Samples should be kept on ice at all times as they warm up considerably during sonication. It is important to leave the samples for at least 1 minute on ice between each round of sonication. Note that sonication parameters will vary according to the sonicator and probe used. It is possible to use a cup-horn sonicator but this requires much longer sonication and it is important to ensure that sonication is even for all samples being sonicated. | ||
*Centrifuge 12 | *Centrifuge 12,000 g, 4 °C, 10 min | ||
'' | ''IP:'' | ||
*Use 800 μl aliquot | *Use 800 μl aliquot for one immunoprecipitation experiment | ||
*Add 20 μl of | *Add 20 μl of a 50% slurry of protein A sepharose or protein A/G beads (note that the beads should be selected to work with the antibody being used) | ||
*Add specific antibody (for example | *Add specific antibody (for example 1 μl of RNA polymerase β subunit; see Material above) | ||
*Incubate at 4 °C | *Incubate at 4 °C overnight on a slow rotator or at room temperature for 90 minutes. Note that most antibodies work fine at room temperature but some require overnight incubation at 4 °C. This has to be determined empirically, as does the amount of antibody required for the IP. | ||
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'''2. Day''' | '''2. Day''' | ||
*Collect each sample on Spin- | *Collect each sample on Spin-X column by centrifugation for 1 min at 3500 rpm | ||
*All following wash steps should be on a rotator at room temperature for 3min with 2 min centrifugation as above: | *All following wash steps should be on a rotator at room temperature for 3min with 2 min centrifugation as above: | ||
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#Wash with 500 μl TE | #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. | *Place filter unit containing the beads into a new 1.5-ml microcentrifuge tube (dolphin-nosed tubes are best because they accommodate the Spin-X column more easily) 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 | *Centrifuge beads 1 min at 3500 rpm, room temperature. Discard filter with beads. Transfer the eluate into new 1.5 ml tube. | ||
beads. Transfer the eluate into new 1.5 ml tube | |||
*Add 80 μl TE and 20 μl Proteinase K to the tube and divide on PCR tubes fitting your PCR machine (for example 2x100μl). | *Add 80 μl TE and 20 μl Proteinase K to the tube and divide on PCR tubes fitting your PCR machine (for example 2x100μl). (Note: many protocols no longer have this step; I get equivalent data when I skip this) | ||
*To reverse cross-links, place tubes into PCR machine. Incubate 2 hr at 42 °C, followed by 6 hr at 65 °C. | *To reverse cross-links, place tubes into PCR machine. Incubate 2 hr at 42 °C, followed by 6 hr at 65 °C. If not using Proteinase K, incubate overnight at 65 °C, or boil samples for 10 minutes. | ||
'''3. Day''' | '''3. Day''' | ||
*Purify DNA by phenol extraction and ethanol precipitation | *Purify DNA by phenol extraction and ethanol precipitation or using a PCR purification kit (e.g. from Qiagen) | ||
*Elute or resuspend in 10-20 ul water | |||
* | *Measure DNA-content ideally 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 | ||
* | * Amplification for microarrays is done using the BioPrime kit (Invitrogen) with dye-coupled dCTP (Cy3 or Cy5 dye), according to the manufacturer's instructions. Typically 5-6 individual ChIP samples are combined for a single amplification/labeling reaction | ||
==Critical steps== | ==Critical steps== | ||
It is important to confirm that sonication results in fragments of a suitable size, i.e. <1 kb on average. This can be done by decrosslinking a sample of crosslinked cell extract, purifying by phenol extraction and ethanol precipitation, RNase treating and running on a gel. | |||
==Troubleshooting== | ==Troubleshooting== |
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