So we have to do process the samples for chip seq and these are the cells of PANC - 1. My colleague fixated these cells 2 months back and we were practicing for running the samples. So we sonicated the cells for 23 cycles with 30 sec pulse on and 1 min pulse off and taking out the 10ul after each time point in the intervals of 2 mins. Yesterday the bands did not came because we forget to purify the DNA because of the purification step so we stored the sonicated sample at 4C and gave 3 more rounds of sonication today and did the purification step as well , we took 10ul sample for the first well and 50ul sample for the other well. We made 1% gel and since the basepair is around 100-250 bp and ran the gel at 110 Volt for 45 mins. Can you guys please tell me what could have gone wrong ? If any additional information is required I can give in the comments.
Why do you expect all fragments to be in the 100 to 250bp range? If you need small fragments, use a suitable restriction enzyme, possibly DpnI or DpnII (depending on the DNA's methylation state) can do the job.
The PANC 1 cells should show the basepairs at 100 - 250bp thats why told that basepair. But what I am getting is nothing but a smear and no bands. We have to check the sonication efficiency thats why we are running the gel to check if the chromatin is sonicated or not. But there is nothing but a smear even though it has 23 - 25 cycles of sonication .
To me, it seems you have fragments of all sizes, and not only 100..250bp. Nothing surprising. Sonication is very difficult to control and to reproduce.
That is ladder of 100bp . The smear is the actual basepair tht shud I have come.
Then it's either not enough material or the DNA has completely disappeared. Either precipitation or degradation, e.g. by DNAses or contaminants.
When storing samples for prolonged times when they shouldn't and repeated.
Try to establish methods before you work with or obtain precious samples.
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