Hey All,
Today we came in removed, stained and viewed our gel. The samples not treated with restriction enzyme showed a similar band, just underneath the wells. This was expected as the samples had not been treated and therefore lysed by the restriction enzymes, so separation according to size did not occur, producing only one/two bands. The samples treated with restriction enzyme showed some separation, but not enough, possibly due to not enough digestion. For both sample types it appeared that the largest plug size used worked the best. Mike also noted that this may be due to a lack of cell density within the plugs. The agarose gel also appeared very messy, and needed to be cleaned up. We destained the gel in TAE buffer, viewed it again and decided on the following modifications for our next PFGE run:
- Run for 20 hours with a 20 second switch interval. The switch interval was decreased in order to produce better/clearer separation.
- 3 large samples for each plug treated with restriction enzyme and those without, as the larger samples seemed to work the best.
- We also deep cleaned our glass wear and autoclaved the buffer used within the sealing agarose, in order to try to clean up our gel as best as possible.
- Finally, when we produce more plugs, we will try to increase the cell density within the plugs and modify our cell suspension buffer/centrifuge stage.
- We are keeping the buffer, ladder, sealing in plugs, temperature and voltage the same.
Excited to see if our modifications produce clearer band separation 🙂