I'm trying to use the Premium RRBS kit (Diagenode) to analyze bovine sperm samples and apparently the restriction enzyme digestion (MspI) is not working for sperm samples, it looks completely undigested. I tried with fibroblast samples, it looks better, but I still have high molecular weight DNA and a weak smear around 100 to 500 bp. Does anyone have experience with this kit or this enzyme for bovine samples? What should I expect after the digestion?
Thank you!
Hello
This article will help you .
https://www.researchgate.net/publication/233422434_Expression_of_KCNQ1OT1_CDKN1C_H19_and_PLAGL1_and_the_methylation_patterns_at_the_KvDMR1_and_H19IGF2_imprinting_control_regions_is_conserved_between_human_and_bovine
Good Luck
Please provide your RE digested gel pics, so it could be better to tell you something
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This is the picture of my gel. I compared to gDNA from fish that looks completely digested.
Please let me know what concentration of enzyme you are using for digestion? even in lane 4 smearing is coming. I would also suggest to make sure that your working reagent should be free from any contamination. you can use 0.5% SDS in loading buffer for dissociation of any enzyme template complex formation.
Hope it will work...Good Luck...
I'm not sure about the enzyme concentration because it's provided by the kit. But the reaction is 1 uL of enzyme, 26 uL of DNA (100 ng) in water and 3 uL of buffer. I tried to double the amount of enzyme but the digested profile was the same. I'm starting to think that this is the profile for bovine digestion at least, because I tried with other bovine cell types (fibroblast and blood cells) and they all look the same.
Thank you for your help!
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