I'm starting a new project with limited expertise in this area. Basically, we're going to use the ChIA-PET technique, which combines ChIP-seq and paired-end-tag library preparation.
I'm working with E. coli, and I need the proteins to stay in-tact when I fragment the DNA, as I will pull down my protein of interest after I fragment the DNA. I know that sonication is usually people's preferred method for DNA fragmentation, but we don't have a sonicator (and new ones are very expensive!). I see that NEB sells a kit with nucleases that cut the DNA randomly in a time-dependent manner. But they recommend having pure DNA when you do the reaction. I was wondering if anyone has tried to use it using crude cell lysate- I'm worried having all that cell gunk will inhibit the reaction. But there's got to be maybe one person who's tried it?
Like I said, this is not my expertise so please feel free to ask me for clarification on something or correct any poor assumptions I am making. Thank you so much!
In case anyone sees this because they are looking for a similar answer... yes, you can use NEB Fragmentase "in situ" to fragment the DNA.
My protocol (super rough, modified from Davis, et al 2011, Methods in Enzymology):
1. Grow 50mL of cells to OD ~ 0.6
2. Fix in 1% formaldehyde for 30 minutes at RT
3. Wash two times in 100mL of PBS
4. Resuspend in IP buffer + protease inhibitors, add Ready-Lyse Lysozyme and 1mg/mL RNase A and treat for 15 minutes at RT
5. Add Fragmentase and incubate at RT for 15-20 minutes
6. Centrifuge at max speed and transfer supernatant to new tube
7. Perform the rest of the IP as in Davis, et al 2011 Methods in Enzymology
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