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Restriction digest band shift?


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#1 miketibs

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Posted 13 March 2013 - 04:15 PM

Hi Everyone,

Having trouble interpreting these results. I amplified a gene through PCR (right lane). The top band was excised and the fragment was purified. I then performed a restriction digest with HINDIII and Xho1 for 1 hour and ran the reaction on the gel (left lane). I'm having problems interpreting why there is a band shift. Is it due to maintained association of the enzymes with the DNA fragment? Would the higher band be undigested DNA and the lower one successfully digested?


Thanks!

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#2 jerryshelly1

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Posted 13 March 2013 - 07:39 PM

The band on the right is your PCR product, which is what you are using as a size control? The band on the left is very similiar in size to your band on the right. I would call them the same product. The presence of your digestion mixture (salts, etc.) could have an affect on the band shift. In the left lane, I would attribute your highest band to a RE association with your PCR product. Easy enough to check, get on your enzymes respective website and heat inactivate your mixture. The band will more than likely disappear. You were right with your conclusions.

Good luck




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