I am optimizing a WGBS protocol trying to ensure my ligation reaction is working. I sonicated my DNA (samples 5-6) and then proceeded with samples 1-4 through end repair, a-tail, and ligation (T4 DNA Ligase) following standard protocol conditions.
To compare both cleaned DNA and also see the adapters, I cleaned 2 of the samples (SPRI beads, 1x ratio) and eluted to the reaction volume (H2O, 40 uL). Samples 1-2 are the cleaned samples. Samples 3-4 are the unclean, still in the reaction buffer. 2% gel, 3 uL EtBr, my loading dye is a 50% glycerol solution added as 1:6 ratio. Gel was run at 90 V for 10 mins, 70 V for 40 mins.
The cleaned and ligated samples compared to the sonicated samples look how they should (~120 bp shift to show that they are ligated, and my recovery is high enough for HSO3- conversion). However, why is the unclean DNA hung up? The bright band at ~120bp I believe is the adapters, but I don't understand why the DNA did not migrate with the rest. Any thoughts? I want to make sure nothing weird is going on before I move on.
T4 DNA ligase (maybe other proteins in this reaction) will interact with DNA. So if you do not purify the ligated product, the DNA will migrate together with ligase ( just like a gel shift assay or EMSA). Nothing weird is going on. To confirm this, you can digest the ligation mix with proteinase K and then run the gel.
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