BAC DNA restriction analysis - I need help :( (Nov/29/2006 )
Since sevral month and even year I am trying to do BAC DNA restriction analysis (of a ~250,000 bps BAC) BUT when I am looking to the results (agarose gel) the DNA fragments are really weak...unclear...It´s awfull So I need urgent help
Usually, I do BAC miniprep (10 ml ON culture, then I elute with 100 µl TE) and digest 50 µl of the miniprep for 5 hours. Then I load it on a 0.8% agarose gel + ethidium bromide and let it run in TBE 1x during 2 days at 100V (day) and 50V (overnight).
Today I tried to stain the gel again with Et.bromide (0.5µg/ml) but it did not improve so much!!
So I hope that someone has the answer of my problem.
Maybe you want to try a BAC midiprep - I regularly do those (I use a commercially available kit with anion exchange chromatography). Putting more DNA in your digests helps, as does stainig the gel in EtBr after the run....
I had the same problem, even doing a maxi bac max from epicentre tech. (200 ml culture)
But i found out that my bac was of poor quality and with contaminants. so i am using now the regular maxi kit of qiagen for bac extraction and I elute at the last step the column with elution buffer warmed at 60 °C.
For the digestion I used like 5 ug of the BAC (that means that around juts 10 digestions can be run from a maxi!) and ran it ON at low voltage in a 0.7% agarose. from this I was able to analyze and extract my bands succesfully.
hope that helped cause i also suffered with those bacs!
well, using bacmids (150kb) i checked presence or absence of my fragment by pcr and sequencing (using MgCl2 adjustment in the pcr).
I didn't even tried restriction analysis...
for your problem, you may have to add etBr in the buffer too, as post staining is less efficient and etbr should be away after 2days of PFGE...
Thanks for your advice...
Well...I already tried to do BAC maxiprep ...it was not better than with miniprep and to be honnest now I have about 10 differents BAC to analyse and I find that´s a bit expensive and lot of work to do those BAC maxiprep
Nevertheless...I will try to increase the DNA level, decrease the agarose%, and add et.bromide in the buffer...I will see what´s happens
Thanks again...I will let you know if there is any progress or not