BAC digestion - (Jun/26/2006 )

Hi all,
Need some help in this one. Has anyone digested BACs for cloning? I have to digest this BAC so I can subclone an 9000 bp fragment (or 5000 bp) in pbluescript. but digestion with two difeferent enzymes just gave me a smear when digesting even up to "45 ug" of DNA
am using EPICENTRE BAC extraction kit and am suspecting my bac concentration is to low even if the real DNA concentratio n is very high (1 ug/ul) could this be just genomic DNA contamination?

I would really appreciate any suggestion

thanks

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hi
i'm not very familiar with BAC, but size is too big for a gel quantitation or even a gel analysis isn't it? i suppose kind of PFGE is better.

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Thanks Fred
This BAC is around 180 Kb and I was expecting a pattern of bands in a common agarose gel 0.8% from which I could extract my band. I can see a band in the undigested BAC but not as strong as I would like to

What is PFGE? can we extarct bands from there?

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The PFGE is the Pulse field gel electrophoresis

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thanks! i would have never guess. can I have better separation thgere? maybe my problem is BAC concentration

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In theory yes but I never did it... I only know something about the principle of PFGE, sorry! I think someone else (maybe fred?) can help!

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hi
I've never done myself PFGE, but saw an experiment of it. The principle allow you to have separation, but recovery of BAC can only be done by electroelution, as for 180kb i don't know any suitable kit

What is your BAC concentration?

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Hi Fred
With this kit from 100 ml culture we were expecting 25 ug (in 200 ul approx... 125 ng/ul) but we end up getting 1 ug/ ul in 200 ul.. that is 200 ug hahah.. I hope that is good
Never tried this electro elution.. do I have to cut a fragment where I expect the bands and put them in the electroelutor?
is that how it works?
Thanks

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Don't know if you figured this out yet but there are a few things you need to consider:
1) the yield you're getting probably represents bacterial chromosomal contamination
a)you can redo your preps making sure to be VERY careful during lysis (invert very gently so as not to shear chromosomal DNA)
b) you can also try cesium chloride prep (though if you don't do a) b) won't help entirely

2) 1) doesn't necessarily matter if you are willing to try the following:
a) you do not need PFGE to isolate 5-9kb fragments-- start with your (10ug) BAC DNA and digest it with the desired enzymes. run it on a gel... don't worry about the "smear", cut out a region in the area of interest based on the DNA ladder you run. gel purify... run the ligation (make sure your cloning vector is appropriately prepared) and transform. Note uncut BAC will get hung up in the well and won't run effectively but 5-10kb frags will run as they normally would
b) because you are looking for a fragment (say 5kb of 180kb) that is about 1:40th of the original DNA molecule you will only get about 200ng of the "right" frag it will be much fainter and may not show as a band (if your BAC is pure of course and w/ contamination the yield of the right frag will go down) Also, because the BAC has many more sites it will be impossible to distinguish this band from others near it and it may indeed look like a smear (alternatively the smear may represent chromosomal contamination)
c) but b doesn't matter if you are willing to screen for your insert by colony PCR. you can do this in a 96 well format and pick 20-50 colonies into your PCR reactions as well as LB-abx and then grow them while you run your PCR. Your size selection, even in the face of bacterial contamination, should yield at least 1-2 colonies but you could do 96 to be safer.
d) I would recommend 1) redoing your preps before starting 2) as your yield will be better.

I have done this to subclone 12.8kb BamHI fragments from a 200kb BAC and was able to successfully pull this off. I started with CsCl pure BAC carefully prepared cut the gel "blind" in a 12-15kb region on the gel and only needed to screen 48 colonies (can't remember how many clones I got but it was more than 5. Hope this helps.

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