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PEG 8000 precipitation/ gel electrophoresis


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#1 hannna

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Posted 19 October 2011 - 06:39 AM

Hi!

So, I just run a gel with the plasmid sample I precipitated with PEG 8000 (washed the pellet with 70% EtOH, and resuspended in TE buffer), and it gives me this weird (high molecular weigh) structures on the gel.

This is the first time I've done PEG 8000 precipitation, so I don't know what's wrong?
Is this some residual PEG that didn't wash off?
Could that affect the ligation I have to do with this product?
Can I get rid of it by doing EtOH precipitation?

I get this pattern repeatedly on my gels. So it's not some artifact from the gel.

Many thanks,


photo:
Size of the left band is 10kb. This should be the size of the right band also, just that it has this weird smearing.

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#2 bob1

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Posted 19 October 2011 - 04:04 PM

Looks like a genomic DNA smear to me.

#3 hannna

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Posted 19 October 2011 - 11:40 PM

It can't be. There was no genomic DNA involved.

It was a 10kb plasmid, cut with 2 restriction enzymes, PEG 8K precipitated, and washed with EtOH. Resuspended in TE.
I did the EtOH washing again. But the smear doesn't go away.

#4 bob1

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Posted 20 October 2011 - 02:49 PM

OK, thought you might have been doing a mini-prep.

I guess it is residual PEG then, or perhaps one of your RE's isn't working properly and you are getting some concatemers.

#5 hannna

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Posted 23 October 2011 - 01:28 PM

Yeah, I guess it's leftover PEG. I can't get rid of it even with multiple EtOH washing.
However, it also seems that it's not affecting my downstream ligation.
Which is good. :)
Thanks!

#6 leelee

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Posted 23 October 2011 - 07:38 PM

It looks to me like you may have pierced the gel with your tip and maybe injected the DNA further back into the gel so you are getting a weird pattern?

But I guess if your ligations are working, and you don't see it again, its just one of those things you can forget about! Posted Image




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