PLasmid extraction: good nanodrop reading but NOTHING on the gel! - (Nov/17/2007 )
Recently i did a plasmid prep on my bacteria Sphingomonas sp . the nanodrop looks very good 69ng/ul and 2.00 ratio but when i ran it on gel nothing showed up, i ran it with another plasmid and the other plasmid was visible,ladder was ok too. It's really confusing. Anybody experienced that before??
p/s --Nothing is wrong with the nanodrop i used a negative control and it turned out ok.
i am suspecting genomic DNA or seomthing at the moment.
Any comments or suggestions would be really appreciated
A ratio of 2.0 shows the presence of RNA not DNA. The ratio for DNA should be 1.8. I would guess that your sample has RNA contamination, and perhaps no DNA. What kind of prep are you doing, and does it involve an RNAse treatment?
I'd guess that your organism has no plasmid.
I concure with phage434. Reading of 2 indicates RNA contamination. How many ul of the sample was loaded into the well?
I concur with phage434. nice . i added 10 microlitre of sample into the well. Should be sufficient i believe. ( > 500ng of DNA or RNA) it should show if there's anything in it. Awesome i don't have plasmid on the wild type!!
I probably need to "shake and bake" my P1 buffer harder when i do the extraction next time to avoid RNA contamination
btw i m using Qiagen mini prep.
have a good turkey day!!
Make sure you added the (separately shipped) RNAse to buffer P1 when you first started using the Qiagen kit.