analysis of DNA using BamH1 and agarose gel electrophoresis - interpretation of results! (Nov/26/2006 )
Hi - i've been analysing solutions of bacterial chromasomal and plasmid DNA by using BamH1 and AGE. I know what the results should show but I am having trouble interpruting what they actually show and why! I've attached the photograph of the gel i obtained from the experiment (sorry if its not too clear).
Lane 1 - lamba Hind III marker
Lane 2 - chromasomal DNA* (undigested)
Lane 3 - chromasomal DNA* (digested with BamH1)
Lane 4 - Plasmid DNA isolated using alkaline lysing method (undigested)
Lane 5 - Plasmid DNA isolated using alkaline lysing method (digested with Bam H1)
Lane 6 - Plasmid DNA isolated using wizard mini prep (undigested)
Lane 7 - Plasmid DNA isolated using wizard mini prep (digested with BamH1)
*isolated using the phenol mixture method. (can give you more info about this method if required)
Thank you very much
First step, focus the camera. It will help if you use a higher F stop (F8 is good) and take a longer exposure to compensate. This makes the focus less sensitive by increasing the depth of field. Are you sure lane 4 is not the digested chromosomal DNA?
in addition focussing correction, your preps are quite badly contaminated with RNA (lanes 2, 3, 4, 5) And to a lesser extend lanes 6, 7.
You probably need to do an addition RNAse step.
Yeah, lane 4 is incorrect for plasmid DNA. Looks like a genomic digest. Please check. Possible mix up between lane 3 and 4.
You probably also need to run a bit more DNA on the digest. The bands look very faint..
I'm sorry about the photo my digital camera isnt great but the actual photo from the experiment is blurred to. I've attached another but dont think its much good.
I'm completely sure lane 4 is not the digested DNA yes. I know the reults look like it is.
is it possible to redo this gel using all the above suggestions?
Because if you want an analysis on the gel (with the assumption that all the lane are good and correct) then you'lll be bombared will plenty of bad news.
eg.. genomic DNA is not clean enough to cut... please clean up with more phenol/chloroform
this is the worst plasmid prep I have seen... it is all degraded.
... strangest effect of Restriction enzyme i have ever seen
.... alll DNA gone... back to the drawing board.
inaddition to a real analysis that the DNA is heavily contaminated with RNA.
For a new comer I would suggest that you spend $ 100 on a Qiagen DNA mini prep kit. This will allow you to get good quality DNA. for further work.