I'm using Agilent HWM DNA extraction kit. PFGE shows a smear starting at around 50kb and going up. However, I want >50kb and preferably 100kb and up. Pippin size select only goes to 40kb. Does anyone know how I can get size select for these large fragment sizes?
I did something similar years ago. I wanted to make a cDNA library, only interested in fragments longer than 1 kb. I ran the sample together with markers in a lower votage for 1 hr. when I was able to see the separation, I cut out the part shorter than 1kb. The remains are all longer than 1kb. Then I ran the gel in a reversed direction for 50 min. All the fragments gathered as a band just beneath the load well. I cut the band and made a library with it. I got what I wanted by this way. Hope that helps.
Tony! Two things come to mind, being naive about the extraction kit you're talking about. 1) A phenol-chloroform extraction, with the ends cut off of pipette tips, can give you large genomic DNA fragments. 2) Using a pulse-field gel electrophoresis may help, but you'd have to look into extracting large fragments from a gel. Sorry I couldn't be of more help!
Jared! Yep, I have fragments starting at 50kb and going up, visualized of course on a pulse-field gel (PFGE). What I want is either an extraction kit that biases toward ultra-high molecular weight DNA (>50bk) or to extract then size select out anything less than 100kb.
Hey Tony,
Aha, the cool science acronyms I don't understand...sorry. I think the phenol-chloroform extraction would be good for biasing towards large fragments, but I don't know of size selection of such large fragments. Sorry, good luck!
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