I am currently working on the creation of an expression-Plasmid for Cas9 in E.coli. I have used the pET303-Vector as a backbone and inserted the Cas9-gene which I cut out of another Plasmid.
My issue is that both Plasmids share an Ampicillin-resistance as well as the same ORI. The unaltered Vector is sized around 5 kilo-bases, the desired expression-Plasmid at roughly 10 kb. So far I have not managed to find a colony that exclusively carries the desired product. Either both Plasmids must have been taken up or the cells did not incorporate the Expression-Plasmid at all. I can see it on the background control of my Colony-PCRs though. I assume the smaller Plasmid eventually kicks the larger out, due to having the same ORI.
I am looking for a way to purify the desired product so this competitive elimination does not occur. I could only come up with using a faster growing E.coli-strain such as Mach-1 instead of XL1_blue and performing a Plasmid-prep as soon as I have a positive clone. I would then linearize via restriction-digest and do a Gel-Extraction of only my Plasmid of interest.
Are there any other ways I could achieve this?
Thank you for taking the time to read and comment :)
Is there a restriction site unique to the undesired plasmid so that you could digest the DNA mixture after ligation and before you transform? Otherwise if you have a mixture of plasmids but you know the plasmid you desire is present in that mixture, you could retransform at very low DNA concentrations (less than 1ng) and test a number of individual transformants, most of them should be transformed by a single plasmid.
I indeed have a site for XhoI in the undesired plasmid that is not present in the final product, I completely overlooked that! I will try digesting my Ligation and using only very little DNA for subsequent transformations.
Thank you so much for the input, Michael J. Benedik :)
I really recommend you to use different vectors with different antibiotic resistance for that work. then you will be able to mange both plasmids through double antibiotic media.
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