I am Using PFGE to screen the presence of larger plasmids above 100 Kb of size from Campylobacters. I came to know that there were plasmids up to the size of 180 kb. Now I want to extract the plasmids so that I could sequence. I tried various methods to extract the plasmids and I even tried gel extraction from PFGE, but still it is very difficult to recover. I would appreciate if anyone has the method to extract these plasmids for sequencing?
I use following BAC isolation kit for large DNA constructs. It works very well
http://www.mn-net.com/tabid/1479/default.aspx
Thanks Babak, Have you use these kits for Campylobacters or some other bacteria? How pure are the plasmids from these kits? I would appreciate your help. Thanks
BAC is a great answer, or you may design your own method if you have enough material. I did isolation of cyanobacterial megaplasmids from PHOGE using BAC but I would bet that guanidin thiocyanate would deliver a cleaner material. I have not played with different concentrations though.
Thanks Tanya...I even tried BAC preparation from Zymo Research and even it failed to show up the megaplasmids. Actually Campylobacters are very difficult to deal with, they are not easy as E. coli. I can see the plasmid in the PFGE gel and I even tried to gel extract it by one of the large fragment gel extraction kit from ZR. Thati s why I am looking for a method which can yield pure Plasmid for sequencing. Thanks
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