Hello everyone!
I am trying to make a library of metagenomic DNA in the plasmid PBlueScript SK+. I fragmented metagenomic DNA by sonication and I confirmed the average size of the fragments is 3kb. I then repaired the DNA fragments with Klenow fragment (NewEngland #EP0054) so that the the ends are blunt. After that, I purified this reapired DNA. Previously, I digested the Plasmid PBScript with EcoRV that also leaves blunt ends. I tried to performed ligation in several Vector:Insert ratios (1:3, 1:1, 3:1...). None of the ratios gave a large number of clones but Ratio 3:1 exhibited the best results in terms of number of colonies and higher proportion of white/blue colonies, which is good .
I extracted plasmid from 15 white colonies of this ligation, I performed Restriction digestion with enzymes XhoI and NotI (that flank EcoRV at both sides in plasmid PBlueScript) so that I could see two bands in agarose gel (1 Plasmid backbone and 2 metagenomic insert). The problem is I obatined a unique band for most of the clones and, moreover, this single band does not match the size of the empty plasmid. Its smaller !!
To me, this result does not make sense. I thought there could be recombination events in the cell but I´m using DH5 alpha strain so it should not happen. Could it be due to an effect of ligation of so long fragments whit blue ends? Am I missing anything?
I have no experience with library construction so any help will be very helpful.
Thanks a lot in advance!
Jorge
It could be a natural plasmid that you extracted along with the metagenomes. In a highly biodiverse sample, it's not out of the question that there would be some plasmids there that have an origin E. coli can read, and the correct resistance gene for them to show up. Of course, they're unlikely to have a natural lacZ, so they're white. It would be difficult to find out for sure, because you don't necessarily know any part of their sequence at all. I'm guessing you don't really care what those white colonies are, as long as you can make your library cloning work.
Blunt end cloning is generally a lousy method. I've seen this work, and not work, off and on with no rhyme or reason myself. I had better luck using a 4-bp overhang at 16C. First, I fragmented the DNA into the right length fragments by partial digest with Sau3AI (needs to get the dosing and timing just right! -- run test gels). And then cloned into the plasmid's BamHI site.
It would be even better if you could do a more modern method like Gibson's Assembly. This would require a PCR step against known degenerate flanking sequences. That works of course only if you have some idea what you're looking for, not for an unbiased library. Or direct ligation of overlaps, which would be a headache in itself.
Probably the Sau3AI / BamHI option was the one I was happiest with overall. I had that working in pSK+ at first, but then stepped it up to cosmid library kit from Epicentre, which uses far longer inserts and was overall much more effective. Got a few 100 billion bp of environmental DNA cloned that way.
I would recommend you change the cloning host. Plasmid rearrangement can occur in dh5 alpha even if it is recA- (that is why people use strains like neb stable and stbl3 for repeats cloning and such) and it has occurred to me multiple times (not in the context of library prep but with much shorter fragment) also the source of the DNA makes a difference certain sequence are known not to be stable in E.coli especially those with skewed GC% contents and repeats. I second John's opinion that you should use cosmids paired with a suitable cloning host.
Thank you very much John Schloendorn and Hanna Alalam
Those were very useful tips and information. I am afraid I cannot use Sau3A + BamHI stretegy since my DNA is already fragmented by sonication with average size of 3kb, which is the size I need. If I "recut" with Sau3A I think I'm going to reduce my sample yo very small fragments (and I cannot start from enviromental sample again because we do not have anymore).
How if I try T/A clonning ? I could dA tail my blunted 3kb fragments, purify them and clone them with a TOPO clonning kit. Does it make sense for you? I know it's expensive and probably the enviromental small plasmid that seems to be around would remain but, hopefully, I will get enough representation of my metagenomic DNA... Do you think this is a good idea?
Thanks a lot in advance again
Jorge
You do not have to change the cloning strategy if you have already preped the sample just change the vector and the host and see if that solves your problems. If you still have problems then then reconsider the strategy.
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