I am new here! My name is Michael, i have 22 years old and i'm on the last year of my biology course.
I found this forum while i was searching for some answers regarding Plasmid DNA extraction, and i though you could give me a little help interpreting the results, as this is my first time with DNA extraction.
The pictures i show bellow are all from the entire class, but what is most interesting, is that although all of us followed the exact protocol, the results were totally different!
The bacteria we used to extract the plasmid DNA were previously grown in LB medium with 5µL ampicilin.
The three buffers we used during the extraction were:
50 mM Tris HCl
100 µg/ml RNase
3M potassium acetate (pH 5,5)
The samples of each class group were run in 1% agarose gel (TAE 1X and 5µL Ethidium bromide).
The sample buffer was composed by
0,2% bromophenol blue
0,2% xylene cianol
60 mM EDTA
Well, and now the worst part, the results (all of the same gel taken on 2 different stages, 2 with white background and 2 in black)
The sample in well number one is the DNA marker. The rest are samples from each student (mine is sample number 3)
So based upon this, i would like to hear your comments.
I am wondering why, in sample 3, i got some bands that are "above" the first band of the marker. IS this normal?
And regarding the bottom huge roudish bands that most people got?
In your opinion, wich sample has more DNA (if any is present)?
And finally, about well number 7, why are the bands so weak?
After this, we also did DNA restriction with 3 enzymes ( Bam
I and Eco
RI. ) and used 10X FastDigest buffer. We used 1 enzime unit, during 30 minutes, at 37ºC (agarose gel at 0,6%)
These are the results we obtained yesterday:
M - Marker (i will post latter wich marker we used, as i do not remember right now)
In the end i will need to build the restriction map to my report, but at this point i am lost. I apologise if any of my questions might sound stupid or inapropriate or if the answer is pretty obvious, but i still need to get used to this and also want to be 100% sure of my conclusions.
Thanks for any help!