Hello Everyone!
Yesterday we ran a 24 hour gel, at 4 V/cm with a 20 second switch interval PFGE gel. We used 3 large restriction enzyme treated plugs and a NEB ladder. Mike also asked us to research the genome for Propionibacterium acnes and the size of the fragments produced when lysed with SpeI. He asked us to do this so that we would be able to sketch a rough drawing of what we would really like to see on a perfect gel. Also to help us look for a more appropriate ladder to use with the Propionibacterium acnes samples, as the average size of a fragment is approx. 100kb, and the NEB yeast we are currently using runs from approx. 1900-225, not quite low enough to compare most fragments.
Today we observed the gel with Mike, which was how he expected it to look, with the NEB bands close together near the top of the gel. However, no bands were visible under the Propionibacterium acnes samples and Mike thinks we really need to have another go at making the plugs, perfecting the cell density and process. We also filmed our latest videoblog!
See you all on Monday 🙂
http://www.neb.uk.com/productcatalogue/productinfotransfer.aspx?id=/New%20England%20Biolabs/Markers%20and%20Ladders/PFG%20Markers/N0340
Also scroll to the bottom where it has ‘Companion Products’
What conclusion did you come to about a more suitable ladder?
We didn’t – except it should range from approx. 50kb-500kb, which would need to be confirmed after a final analysis of the size of all fragments. We searched around last night but i wasn’t sure on anything. Think we need to look over some with you, if you don’t mind?