How to determine success of a digest producing short segment

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faithrobert
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How to determine success of a digest producing short segment

Post by faithrobert » Fri Sep 10, 2010 8:44 pm

Hi All,
I have a plasmid of 4.008 kb which I have digested with BamHI and NotI concurrently. The digestion is supposed to produce two segments of of 45 bp and 3953 bp respectively. I ran the digestion on 1.5% gel but there was no obvious difference between the linearised vector and the doubly digested one. Also the 45 bp segment was not visible on the gel despite loading 20 ul of reaction. On the assumption that the double digest worked, I used the 3953 bp segment for a ligation reaction after using SureClean kit to clean up. I have screened 50 colonies and not a single clone had my insert. I think that the problem may either be with my ligation reactions or that my double restriction digest didnt really work.
How would I verify that my double digest actually worked?

regards

Faith

MJD
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Re: How to determine success of a digest producing short segment

Post by MJD » Fri Sep 17, 2010 2:13 pm

You likely ran off your 45bp segment of the 1.5% agarose gel. You might consider running a more concentrated gel like a 3.5 agarose gel to resolve the 45bp segment and the apparent difference between the linearized vector and the doubly digested linearized vector. I suggest you also run the undigested vector as a control it should run higher than the linearized vector. Make sure you have a DNA ladder that has a 100bp marker otherwise you might order one for less an hundred dollars. NEB has one a nice 2-log DNA ladder (10kb-100bp).
You are sure to either get separation of the 4008 kb linearized vector and 3953kb segment or hopefully the 45kb fragment will show up, but be sure not to run this band off the gel. You may want to try performing the digestion in duplicate in case you get some degradation of the sample. Running the undigested vector as a control will show the apparent difference between the linearized vector and the uncut vector. Good Luck!

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JackBean
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Post by JackBean » Fri Sep 24, 2010 7:09 am

You should run it in more concentrated agarose, because differentiate 4008 and 3953 bp is quite hard.

Or find some other double digest
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

testsubjectzz
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Post by testsubjectzz » Sat Sep 25, 2010 1:43 pm

I would say it most likely did run off on a 1.5% gel, if it were me trying that I would try a 5% agarose gel also @jackbean for big segments of dna it is best to use a low % gel instead of a high one.

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JackBean
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Post by JackBean » Sat Sep 25, 2010 2:59 pm

sure, did I said something else? :roll:
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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