SEA-PHAGES | Gordonia DNA not cutting

Link to this post \| posted 19 Nov, 2020 16:12
asprenkle	Anyone have any trouble getting a good RE digest of Gordonia DNA? I've got good DNA from M. foliorum, and we're trying Gordonia rubripertincta for the first time this year. I've noticed it's a bit waxier than M. foliorum. I'm getting OK DNA concentrations with a new Wizard kit, but my Mf DNA cuts where my Gr does not. Any tips/tricks I'm missing? Thanks!

Link to this post \| posted 19 Nov, 2020 16:58
viknesh	Hi Amy, Would you mind sharing a little bit more information about what you're using and seeing? For example: What restriction enzymes are you using? Did you also include DNA for your control phage? If so, what phage is it? Chidiebere? Could you share a gel image of what you are seeing? Thanks. Vic asprenkle Anyone have any trouble getting a good RE digest of Gordonia DNA? I've got good DNA from M. foliorum, and we're trying Gordonia rubripertincta for the first time this year. I've noticed it's a bit waxier than M. foliorum. I'm getting OK DNA concentrations with a new Wizard kit, but my Mf DNA cuts where my Gr does not. Any tips/tricks I'm missing? Thanks!

Link to this post | posted 19 Nov, 2020 16:58

viknesh

Hi Amy,

Would you mind sharing a little bit more information about what you're using and seeing? For example:

What restriction enzymes are you using? Did you also include DNA for your control phage? If so, what phage is it? Chidiebere? Could you share a gel image of what you are seeing?
Thanks.
Vic

asprenkle
Anyone have any trouble getting a good RE digest of Gordonia DNA? I've got good DNA from M. foliorum, and we're trying Gordonia rubripertincta for the first time this year. I've noticed it's a bit waxier than M. foliorum. I'm getting OK DNA concentrations with a new Wizard kit, but my Mf DNA cuts where my Gr does not. Any tips/tricks I'm missing?
Thanks!

Link to this post \| posted 19 Nov, 2020 18:06
erin.windsor	The only enzyme that cuts well for us in Gordonia rubripertincta is NspI. See attached. 245Kb

Link to this post \| posted 19 Nov, 2020 18:14
viknesh	erin.windsor The only enzyme that cuts well for us in Gordonia rubripertincta is NspI. See attached. Thanks for sharing this, Erin. We too have found that NspI reliably cuts DNA of phages isolated on G. rubripertincta. We've also found that BamHI, HindIII, and SacII also cut a few of the G. rubripertincta phages we've isolated. Your results will defintiely help us get a better grasp of which enzymes are best to recommend moving forward. Vic

Link to this post | posted 19 Nov, 2020 18:14

viknesh

erin.windsor
The only enzyme that cuts well for us in Gordonia rubripertincta is NspI. See attached.

Thanks for sharing this, Erin. We too have found that NspI reliably cuts DNA of phages isolated on G. rubripertincta. We've also found that BamHI, HindIII, and SacII also cut a few of the G. rubripertincta phages we've isolated. Your results will defintiely help us get a better grasp of which enzymes are best to recommend moving forward.

Vic

Link to this post \| posted 30 Nov, 2020 14:25
asprenkle	I was finally able to get cutting, and with NspI but not HindIII. I did not use other enzymes after Dan suggested he only needed to see UC DNA, so I was being frugal with enzymes and gel lanes. I had omitted the 65C incubation after DNA isolation. That is clearly a necessary step Thanks for your help!

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Gordonia DNA not cutting