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insert release not as intense as the plasmid


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3 replies to this topic

#1 Curtis

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Posted 09 March 2010 - 06:14 AM

Hi I cloned my PCR product into pJet vector and after that I digested and ran it on gel to make sure the insert is there. The released product doesn't look as intense as the plasmid itself? why is that? is it always like that?

lane 1: ladder
lane 2: single digested plasmid + insert
lane 3: double digested plasmid + insert

plasmid almost 4kb
insert almost 1kb

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#2 jonny_boy

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Posted 09 March 2010 - 06:36 AM

Short answer is yes! It is to do with the amount of EtBr incorporation. A larger product will always look more intense than a smaller one present at a 1:1 amount as it will incorporate more ethidium and therefore produce more fluorescence under UV.

Hope that helps!

#3 phage434

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Posted 09 March 2010 - 06:38 AM

It's always like that. The intensity of the band is proportional to the amount of DNA. Short fragments of DNA have less DNA (weight) per molecule. Since there are the same number of each fragment type (you started with single molecules), the intensity of the shorter bands will be less.

#4 HomeBrew

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Posted 09 March 2010 - 12:28 PM

In fact, if it's not like that, you need to suspect your insert is actually a concatemer, containing multiple copies of your intended insert in series...




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