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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?

#4 OldCloner

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Posted 13 June 2018 - 06:35 AM

You said, "after growing and plasmid prep of the selected transformant, the plasmid dna was run on gel."  Was that undigested? It's hard to judge correct size of undigested plasmids.

 

It sounds like you only picked one colony after doing the colony-PCR screening.  If you had more positives, it would have been a good idea to pick several, maybe 3-4 at least, for further analysis.  It is comforting to know you got more than one of your products and they all look alike, digest the same, etc. It is worth the extra reagents. Sometimes one is just plain odd, but the others will look OK. Then you can dismiss the odd one as an accident of cloning (weird stuff happens).  If you only had one positive by PCR, it pays to be a bit suspicious (voice of experience).






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