plasmid isolation and gel - (Oct/21/2008 )
Hello there,
perhaps you have some ideas on my plasmid isolation?
I isolated my pET28-plasmid with insert from E.coli BL21 (DE3) Gold cells (endA-). It should have a size of about 6.5 kb. On my agarose gel I see a band between 7 and 8 kb, but nearer to 8 kb. Could this be my plasmid, maybe in a nicked form? Unfortunately I can't show you the picture. I used the kit from Qiagen.
I also have a very low amount of plasmid (43 ng per mikroliter). How can I increase it? I used about 4.5 ml of culture.
Thank you very much
-sonnenstern1-
QUOTE (sonnenstern1 @ Oct 21 2008, 05:08 AM)
Hello there,
perhaps you have some ideas on my plasmid isolation?
I isolated my pET28-plasmid with insert from E.coli BL21 (DE3) Gold cells (endA-). It should have a size of about 6.5 kb. On my agarose gel I see a band between 7 and 8 kb, but nearer to 8 kb. Could this be my plasmid, maybe in a nicked form? Unfortunately I can't show you the picture. I used the kit from Qiagen.
I also have a very low amount of plasmid (43 ng per mikroliter). How can I increase it? I used about 4.5 ml of culture.
Thank you very much
perhaps you have some ideas on my plasmid isolation?
I isolated my pET28-plasmid with insert from E.coli BL21 (DE3) Gold cells (endA-). It should have a size of about 6.5 kb. On my agarose gel I see a band between 7 and 8 kb, but nearer to 8 kb. Could this be my plasmid, maybe in a nicked form? Unfortunately I can't show you the picture. I used the kit from Qiagen.
I also have a very low amount of plasmid (43 ng per mikroliter). How can I increase it? I used about 4.5 ml of culture.
Thank you very much
It's likely that your plasmid is supercoiled on the gel and so it will look like it is running higher. You could try digesting it with something that cuts once in the plasmid to make it linear and therefore see the true size of the plasmid. To increase the concentration from your plasmid prep, heat up the elution buffer to 55C before using it.
-smu2-