Introduction to ChIP webinar
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Our resident ChIP expert, Dr. Karen Halls, presented a webinar on an ‘Introduction to ChIP, Principles and Troubleshooting’.
Read the most common ChIP questions, and their answers of course.
- Does formaldehyde fixation need optimization?
- What type of ChIP is right for me?
- Is MNase digestion possible in X-ChIP?
- How long are your sonication cycles?
- Guidance on how to find promoter sequences for histone ChIP
- What are the best positive control genes and regions?
- Do you have any resources for identifiying sequence for ChIP primer design?
- Why is the chromatin fragment ideal size between 150 - 1000bp?
- How is chromatin or DNA value assessed?
- How do you prepare tissue for ChIP?
- Are there any recommendations when using a probe sonicator for the sonication step?
- If I use native ChIP, do I still need sonication?
The time of fixation with formaldehyde needs optimization by the researcher. Is that true because 2-30 min is a long time?
Answer: Generally this doesn’t need to be optimised, 10 minutes should be sufficient for cell culture or slightly longer for tissue samples.
If I am planning to study epigenetic modification especially for detection histone modification, what is the most suitable technique, X-chip or native one?
Answer: X-ChIP or N-ChIP would work well. We have more experience of X-ChIP so would probably recommend this technique. This tends to be a more straightforward method to get right.
Answer: Yes, this is possible but it will not be as effective as sonication due to the protein/protein cross-linking. Some areas will not digest very efficiently. If you would like to perform enzymatic digestion, we would suggest cross-linking for reduced time and including an additional short sonication step to ensure DNA is fragmented sufficiently.
In your example of sonication optimization, how long are your sonication cycles? I presume you do not sonicate constantly for 20 seconds?
Answer: It was a 1 minute cycle with 30 seconds sonication, 30 seconds without, for 20 minutes.
Can you provide guidance on how to find promoter sequences for histone ChIP, and how to design primers that are specific to promoters that have modified histones bound to them?
We are doing ChIPs for histone modifications in our genes of interest. How do we identify good positive control genes and regions for designing our test primers, when histone marks are highly variable by cell type, and by publications?
Is there a website besides the University of California, Santa Cruz genome browser which is a reliable resource for identifying sequence for ChIP primer design?
Answer to the last three questions: As far as we are aware there is no database which shows all histone modifications across all genomic locations (this will vary by cell type and cell line). The following two links might help:
»" rel="nofollow" target="_blank">http://www.bioinformatics2.wsu.edu/cgi-bin/ChromatinDB/cgi/visualize_select.pl">» ChromatinDB: A database of genome-wide histone modification patterns for Saccharomyces cerevisiae
For positive control genes, we would suggest trying some of the standard histone marks for gene activation and silencing and check the literature. There are websites and software that will assist with primer design. There will be software provided with real-time PCR machines. We have used Biotools" rel="nofollow" target="_blank">http://biotools.umassmed.edu/bioapps/primer3_www.cgi">Biotools primer tool 3 in the past, but others may be available.
Answer: N-ChIP will be ~150 bp, X-ChIP will be ~500 bp. It must be larger for X-ChIP to ensure that intra nucleosomal DNA is not fragmented and nucleosomes lost
Answer: 25 ug of chromatin, this can be measured by Bradford assay. Use ~1x106 cells per ChIP sample
What is the procedure for preparing tissue from mouse brain? For example, do you perfuse the mouse with 1.5% PFA and then micro dissect the region of interest? Is there a minimal volume of tissue needed for detection?
Answer: We recommend to check out our ChIP tissue protocol for more information.
Are there any recommendations when using a probe sonicator for the sonication step? I feel like I'm not getting very consistency in my sonications
Answer: It is very difficult to obtain consistency with a sonicating probe. We would recommend to sonicate for very short bursts and rest the sample on ice. Foaming is the major problem when using a probe.
We would recommend a sonicating waterbath, as it provides reproducible results and minimzes sample foaming.
Answer: No it is not necessary to sonicate. Micrococcal nuclease digestion will be sufficient.
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