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Plasmid dna size


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#1 Angeline

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Posted 01 December 2017 - 04:10 AM

I clone a 700 bp insert into a 10kb vector. Prior to transformation, a double digest of the vector and pcr product was done. A gel purification was after double digest and I am certain I cut out the correct product. With the gel purified insert and vector I did an overnight ligation and later transformed the ligation product into competent cells.
Colony pcr was done to select the positive transformants. after growing and plasmid prep of the selected transformant, the plasmid dna was run on gel. My expected product size would be more than 10k. However, apart on a band at more than 10 kb, a band was also obtained at around 1.5 kb. I couldn?t find an explanation towards the 1.5 kb product. I appreciate help here for possible explanation towards such result. Am I getting the correct product? Thank you.

#2 bob1

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Posted 01 December 2017 - 07:06 AM

There are a couple of things that could be going wrong - 

 

It could  be that you are getting fragmentation of the plasmid during isolation, but this would come out as a smear usually.

 

It might be that the bacterial strain you are using has more than one plasmid in it. Try digesting the plasmid prep with a couple of RE's (usually you would use the ones you used in the cloning) to see if this band disappears.



#3 Angeline

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Posted 02 December 2017 - 03:37 AM

Thank you very much for the suggestion. I?m just wondering, how would it be possible to have 2 plasmid in the bacterial cells? Means there?s contamination some where?




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