Is the DNA from cells or tissue? The purpose of embedding in LGT agarose is to minimise shearing. A block of LGT agarose with embedded cells can be digested o/n with PK,SDS RNase etc. I used to use FIGE of virally infected cells to separate 180kDa viral DNA from total human genomic DNA quite successfully. I used to add the PK into the LGT agar + EDTA, allow to set and put in digestion buffer with SDS + PK to get a gentle lysis. As you are not trying to separate DNA species, can you just put your digested block into a small dialysis chamber and electrophorese out the DNA. Alternatively you could do the whole digest in a 100 kDa cut off Float-a-lyzer and slowly dialyse out the digested proteins. Your pipetting will unfortunatelyy introduce some element of shearing.

Just a thought.

Ian Teo

Years ago people also used to use high speed density gradient centrifugation

Ian

Thanks for your answer and the float-a-lyser suggestion (didnt know this before).

The DNA needs to be extracted from whole insects. I agree that any pipetting is increasing the shearing ... and thats what i need to avoid to have in the end 500kb to 1 Mb (or longer) DNA pieces. How much is 180 kDa DNA? I'm not familiar to have the DNA size in this measure. I found a conversion which says 1basepair DNA is about 650 dalton, which would mean 270 bp for the viral DNA. Or do you mean DNA coding for a 180kDa Protein. then its almost 5kb DNA (10kDa Protein = 270 bp DNA).

The float-a-lyser looks interesting. I will check if its possible for our purpose.

I also considered this gradient centrifugation. But from what i was reading in older papers, the length of the purified DNA is too short (only some 10kb) and doesnt reach up to 500kb or more.

Would you think a very careful phenol-chloroform extraction could work, if i use petridishes (large surface for phasing) and old style pipettors (larger pipet diameter). Or is this still too much friction for the DNA?

my error - I meant 160kbp (HHV-6 genome) Wasn't thinking straight. Gradient centrifugation was usually used to purify viral DNA and plasmid DNA which is obviously smaller - the genomic DNA went to the bottom of the tube - one would have to play about with densities of sucrose , CsCl etc to get a band, but it would separate away the very large from smaller stuff. The only problem with the phenol chloroform is that it would involve some measure of mixing. I used to work in the field of DNA repair. We would lyse radio-labelled cells on top of a Sucrose gradient, centrifuge and collect fractions to get a measure of ds DNA breaks - old tech, though I cant tell you what size our fragments were. Are you trying to make artificial x-somes? library?

Ian

Nice, i will check the gradient and if it could be sufficient by modifying the parameters (the papers i checked on this before had smaller size, too small fragments for my needs).

Its for optical mapping using those nanopore things, work up to 500kb at the moment and would help big time for genome assembly and structural analyses

As suggested by @Ian Teo, producing agarose plugs with embeded DNA is not so complex.

But do you realy need DNA over 500 kb? If your DNA is for Optical Mapping, the size of the molecules is not a concern. The DNA is usually digested using restriction endonucleases and the consecutive fragments are further aligned with fragments from other molecules using appropriate softwares.

I agree with Ian and Frédéric - embedding whole cells in agarose plugs and lysing is super easy and will effectively recover entire bacterial (≥5 Mbp) and eukaryotic chromosomes. This is where Bio-Rad gets some of their PFGE standards from (http://www.bio-rad.com/en-us/product/pulsed-field-standards). Getting the whole chromosomes out of the agarose in one piece is tricky and requires something like beta-agarase (https://www.neb.com/products/m0392-agarase-i) or electroelution (http://www.jove.com/video/2136/electroeluting-dna-fragments).

However, these techniques do allow you to isolate specific chromosomes after running them out by PFGE. I did this to extract a 856kb bacterial chromosome once upon a time for a small insert shotgun clone library.