Hello Everyone!
Came into the lab today, removed, stained and viewed our latest gel. We observed a clear similar band under all the samples not treated with restriction enzyme, and slightly blurry similar band under all samples treated with restriction enzyme. Although the position of the bands are correct, the blurriness suggests that separation is still not occurring properly. There also appears to be a problem with the sealing agarose, resulting in very dark wells as DNA cannot properly leave the wells. This was actually my fault, as when making up the sealing agarose for the wells, i had been mixing the same 1g of agarose used in making the actual gel, in the smaller amount of buffer (50ml) used when making the sealing agarose, instead of halving the agarose to 0.5g. Therefore we were attempting to seal the wells with 2% agarose, which explains the difficulty we had in pipetting it (the formation of bubbles), the dark wells and smearing of samples through the gel.
We decided on the following modifications:
- Increase the agarose gel % from 1% to 1.3/1.4%, in order to help increase the separation of fragments.
- Use the correct agarose gel % to seal the plugs into the wells, hopefully decreasing the dark wells and smearing, and increasing the movement of DNA through the gel clearly.
We then ran a standard 0.8% agarose gel, with 0.5X TAE buffer, for 2 hours at 100V. We ran 3 large samples of restriction enzyme treated plugs, 1 Kb ladder and lambda Hind III ladder. We sealed the plugs in with 0.8% agarose (0.4g agarose and 50ml 0.5X TAE buffer).
The results of this confirmed our modifications, and tomorrow we will be running a new PFGE gel for 24 hours, at 4 V/cm with a 20 second switch interval, applying our modifications.
See you all tomorrow 🙂