Hello fellow researchers, I did a simple CUT&TAG for H3K27me3 in HEK293T cells. I checked my results using PCR. If you look at this picture that I attached, I first thought I had untagmented DNA bands in my samples. However, this doesn't explain the band in the IgG sample which is the first lane after the marker. Because, if it's untagmented genomic DNA, it's in every sample including IgG regardless of antibody treatment. Any insights for troubleshooting will be helpful.
The IgG sample may contain DNA. The PCR reaction may not be completely specific or there may be contamination in the PCR reaction. Use more specific primers and keep the lab sterile.
It is not uncommon to observe a band in the negative control (IgG) for CUT&TAG PCR. This could be due to several reasons, including:
It is important to carefully examine the experimental protocol and troubleshoot each step to identify the source of the band in the negative control.
These video playlists might be helpful to you:
https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE
https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw
https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU
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