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I've run out of ideas.... Restriction enzyme digestion


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#1 moerae

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Posted 31 January 2012 - 06:36 PM

So after my plasmid problem... I've hit a restriction digestion problem... *sigh* If it's not one thing it's something else. Before when I was cutting my plasmids (I had to concentrate it down using the speedivac, my plasmids are in TE buffer) with BssHII, I ran it on a gel to check whether digestion was complete and found multiple smeary bands. This has never happened to me before when I've used this enzyme and for plasmids that share a similar backbone to the one I'm currently working with. I thought it was because there was too much salt concentrated in the solution from the TE causing problems so I've eluted a new batch in water and concentrated it down. Set up another reaction and it doesn't appear that BssHII is cutting... which it should. I am using the correct plasmid... so I have no idea what's going on.

Does concentrated TE cause RE cutting problems?

#2 scolix

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Posted 01 February 2012 - 12:25 AM

get a new vial of the enzyme. Try to have the DNA in water and digest in 50ul reaction volume. Its also possible tha the DNA is degrading, leading to your smeary bands

#3 mdfenko

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Posted 01 February 2012 - 09:26 AM

Does concentrated TE cause RE cutting problems?

it may. you also concentrate edta which will chelate magnesium. your free magnesium concentration will be lower than required.
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#4 moerae

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Posted 01 February 2012 - 02:09 PM

I've tried cutting my two plasmids with a different enzyme and one of them has worked (so that's half of it)... The other one still appears to be smeary and horrible. My plasmid preps are fresh so would be DNA be degraded in these ones?

#5 mdfenko

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Posted 02 February 2012 - 06:46 AM

you may be seeing "star" activity with the re that gives you smears. you may want to try to try another re if there is a site. otherwise you'll have to try to optimize the conditions for the original re.
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#6 moerae

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Posted 02 February 2012 - 11:00 AM

Ok... If it was exhibiting star activity shouldn't it also affect the other plamid that worked?

#7 mdfenko

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Posted 03 February 2012 - 07:20 AM

Ok... If it was exhibiting star activity shouldn't it also affect the other plamid that worked?

not necessarily. all of the conditions are not the same (ie, higher concentration of edta present, pH may be off due to increased amount of tris).
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