I am mystified. It does somewhat looks that it's got some fuzzy bands that could correspond to some non-certain binding the thing is from the sample lanes, nonetheless it doesn't look like spillover or any trivial purpose.
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To be able to make an effort to carry you a simple (but in actual fact too simplistic) response struggling with a a lot more than intricate scenario, I would suggest:
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 (one Plasmid backbone and a couple of metagenomic insert).
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I'm trying to make a library of metagenomic DNA while in the plasmid PBlueScript SK+. I fragmented metagenomic DNA by sonication And that i confirmed the normal dimension of your 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.
Nonetheless, should you have two isogenic cell strains that vary in one or other DNA repair function/s, comparing CFA success after cytotoxin publicity should reveal thepossible purpose from the faulty repair function during the resistance to that individual drug.
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