Has anyone done ChIP experiments in Sorghum? I would like to know the optimum conditions say for example the formaldehyde percentage, optimum sonication conditions and the percentage of triton-X100, etc. to get good starting material... any help in this experiment is appreciated.
New Stock of 37% Formaldehyde (methanol free) should be used to make 1% formaldehyde for 20 min fixation for tissues. I don't know about sorghum. Sonication conditions depend on tissue or sample type. The basic principle 20-30 sec pulse time and 30 sec rest for 1 hr at 4oC should give you good results. Once you add cell lysis buffer, you need to pass the sample at least 20 times through 23 gauge need, so that DNA get sheared and then perform sonication as above.
Good luck
Rajendra
Thanks! I have done my experiments with 0.1%, 1% formaldehyde with 10' and 30' sonication time, however I don't get any DNA band as such, looks like a broad spectrum on 1% agarose gel. But I haven't tried passing the sample 20 times through 23 gauge needle so may be I will give a try to this!! Thanks Again!
As with any experiment ChIP protocol also needs standardisation in your crop. Only to your help can be any literature showing ChIP in sorghum (as a starting guideline). Otherwise see if somebody has isolated mononucleosomes in sorghum so that you get an idea for fixation. Additionally, you also remember that a good sonication does not always guarantee for good ChIP, if you have fragmented DNA using long pulses a second possibility is that protein DNA interactions have also got broken. So be carefull and watchful. Yes, CHIP exptt in maize can also be of help as well because both maize and sorghum plant have similar physiology and hence may give you a good start point and for this a paper that i may suggest is attached here with( perhapsyou may well have this already but i can be no more of help, sorry.........). Good Luck for your exptt.
- Badges
- Science topic
- Similar topics
- Biological Science
- Botany
- Plant Biology