To test if my double digest of the pTriex-3 vector is working I ran my 'uncut plasmid', 'double digested plasmid' and 'enzyme 1 cut plasmid' and 'enzyme 2 cut plasmid' on an agarose gel.
Double digested and single digested looks the same. Indicating both enzymes were able to cut the plasmid. The digested band falls in the middle of the 2 bands that are present in the uncut plasmid lane. Which I believe would be nicked plasmid higher up and super coiled plasmid lower down.
The problem I am having is that my linear plasmid band is higher on the gel than it should be. Ptriex should be 5 kbp but it instead looks 10.
I cant explain this discrepency? I thought perhaps it might be overloaded but I ran the same amount of DNA in all lanes ~50g and the digested insert has run to the correct size. The buffers for both insert and vector digestion were the same.
I should add that the agarose gel had gelred added to it and the laoding dye used to laod the dna samples into the wells had gelred added
The ORF of the plasmid has been sequenced and is correct so I am certain it is the correct plasmid. Ill try add a picture later
Marker
Insert
Double Digest plasmid
Double Digest plasmid repeat
Single Digest plasmid Enzyme 1
Single DIgest plasmid Ensyme 2
Uncut Plasmid
Marker
Hi, Gary.You use too high amperage. In this case is hard to see the proper length of the fragments.
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