Expt: I have isolated genomic DNA from LNCaP cells and would like to crosslink a small molecule (Chem-seq) to the DNA. The small molecule is an alkylator so it will crosslink DNA on its own. The buffer I am using for the crosslinking is TKMC (10 mM Tris-HCl [pH 7], 10 mM KCl, 10 mM MgCl2, 5 mM CaCl2). After crosslinking I want to fragment the genome to 100 - 600 bp (300-400 bp average). I am using a Bioruptor 300.
Question: Should the TKMC buffer be sufficient for sonication? Most references I have found use TE buffer (pH 7.5 - 8), and I can't find a reference using TKMC.
More info: I tried a direct comparison between the two buffers (TKMC v TE pH8) and the TE buffer seems to fragment to smaller sizes compared to the TKMC buffer.
For example: Biorupter 300, Low setting, 1 cycle = 30/30 ON/OFF @ 3 C, 5 ng/uL DNA in 300 uL
average frag size @ 30 cycles: TE = 350bp, TKMC = 500bp
Any suggestions? Using TE buffer for my alkylation crosslinking would not work due to presence of EDTA. Should I play around with the cycle ON/OFF times?
Any help would be awesome! Thanks.
After running a comparison between TKMC, TKMC + 1mM EDTA, TKMC + 10 mM EDTA there was a slight tightening up of the fragment range as I added EDTA. In addition, the fragment range (sizes) was smaller when EDTA was added (compared to TKMC w/o EDTA, after the same number of cycles). The results were not drastic but there was indeed a noticeable effect on fragment size and range. I will proceed with adding EDTA before sonication. Thank you for your suggestions!