I tried to apply 3C experiment in NIH/3T3 cell line. NIH/3T3 is a Mouse Embryonic Fibroblast cell line.
1*10^7 cells were used for 3C experiment. However, by adding 0.3% sds in 1* NEB buffer 2.1, the nuclei were hardly lysised when incubating at 37 °C. I also tired to increase sds concentration to 1% and incubate at 65 °C for half an hour, but still not working.
How can I lysis the nuclei?
http://www.ncbi.nlm.nih.gov/pubmed/17641637
usually you can obtain the nuclear membrane to dissolve using hypothonic solutions, but I am not sure it will work with this protocol
Hi Yiquan,
If your cell lysis is incomplete, this step will work poorly. Make sure that your cells are dissociated /not agregated prior to step 5 in the protocol (adding of 5 ml cold lysis buffer). I use a cell strainer for all of my cells. After the cell lysis, confirm that indeed you only have nuclei left. If the cell lysis is not efficient, you might also want to try a dounce homogenizer.
If the cells are well lysed, you may see that the nuclei still agregate when adding the restriction buffer. This aggregation should disappear after a few mins (5-10) at 60 degrees in the presence of SDS. If not, your samples will not work. Do not keep the cells at high temperature for a long time (30 mins), because you will damage the cross-links.
Good luck,
Daan
There is a detergent-free commercial kit for isolation of nuclei:
https://inventbiotech.com/products/minute-detergent-free-nuclei-isolation-kit?_pos=1&_sid=3e52cfd28&_ss=r
Traditional methods use detergent to open up the cells, causing potential leakage and aggregation of nuclei.
- Badges
- Science topic
- Similar topics
- Methodology
- Laboratory
- Cellular Biology Techniques