Why dont you try Qiagen BAC DNA isolation kit. It gives very high yield in ug and better purified DNA which can be used for sequencing.

Hi, we have very good experience with NucleoBond BAC 100 . Around 100 micrograms from 500 ml cultures.

I suggest you to use Qiagen Midiprep or Large-construct kits (depending if you want to remove E. coli genome or not) for high quality BAC DNA isolation, following manufacturer's recommendations for low-copy plasmids.

Hi, Abigail

I recommand you to try BAC DNA MiniPrep Kit or ZR BAC DNA Miniprep Kit. Find some details of process in these links:

-http://www.geneflow.co/pdfs/18050%20BAC%20DNA%20MiniPrep%20Kit%20Info.pdf

-https://www.zymoresearch.com/dna/bac-yac-pac-dna-purification/zr-bac-dna-miniprep-kit

Thank you

Marius

I also recommend Macherey-Nagel NucleoBond BAC 100 or Xtra BAC Kits. These have usually had better and more consistent yields than Quiagen Large-Construct Kit.

Zymo BAC miniprep columns are very sensitive to bacterial culture size used and do not give good yields consistently. If your application does not require 100ug yields you could use regular miniprep protocol from 5ml culture with 400ug/ml RNase A in Solution 1 and do the phenol:chlorophorm + ethanol precipitation as you are currently doing.

Good luck

I have used the nucleobond extra midi kits for years and have found these columns to be just as good as any other kit designed for BACs but at less than half the cost (we get them at ~$8 a column). For any BAC you need to start with 100ml or more of culture to get ug amounts of DNA since they are low copy plasmids. In line with what is said above, we routinely get about 25 ug of DNA from 100 ml culture.

1 - Use RNAse and some more.  Be generous with your RNAse.

RNA presents about half of the nuclei acid content of the cell. To remove RNA, the easiest way to do so is to add RNAse. More is better. As for columns... the column will bind both RNA and DNA, with RNA being eluted from the column during the wash steps. It should be noted that column have a maximum binding capacity. So if you want as much DNA out of your column, one way is remove as much RNA as possible before binding.

2 - Reuse the column.

After the DNA is eluted from the column, You can reuse the column by reequlibrating it with Equlibrating buffer. So split your sample into two. And go through two rounds of Column equilibration - DNA binding-washing-elutions. You can get more DNA that way.

3. Grow more cells!

BACs are single copy. You won't be getting ug of DNA from 5ml culture. You need 100ml, 500ml even 1L cultures to get ug DNA.

4.Grow your cells slowly.

If your insert has many repeats... or makes your cell unhappy, You will need grow your cells slowly... for BAC work definitely use low salt LB (5g NaCl/ liter), and you may want to use O2 limiting conditions (ie tape around the foil to limit aeration) .  Growing longer than 16hr may also be required. Also drop the Cm concentration, use 12.5ug/ml. Your are using too high Cm concentration for BAC.

5. Cell line.

You want to use a recombination minus e coli line. DH10B is the cheapest. Stbl4 is a bit better. Avoid using e coli lines used for protein expression

I always use the Genopure Plasmid Maxi Kit from Roche.

I start with 500 ml overnight cultures and do the low copy nbr protocol with elution buffer at 55 degrees.

I usually get loads of BAC DNA. More than enough to prepare my injection fragments.

Thanks for everyone's input!

I happened to have the Mecherey Nagel Nucleobond Extra Midi kit in lab, but I never thought to try it since I'm used to column kits having a cutoff of ~25kb. Turns out this one is supposed to work up to 300kb, you just have to scale up the culture and use more of the reagents. I used ~350mL of culture and it worked very nicely, although I didn't anticipate how incredibly viscous and difficult to pipette such a high MW product might be once I had it. But my digest gave the expected product, so all is well. Thanks again, everyone!