I am trying to do Gibson assembly. After ligating insert with backbone I have transformed the ligated plasmid in DH5alpha via electroporation. After transformation I got some colonies in Luria agar plate containing selective antibiotic and have done colony pcr from those colonies and got band at desired size from some colonies. For doing colony pcr I have designed forward primer from plasmid region and reverse primer from insert region. Then I have done replica plating from positive colonies and again done colony pcr and I have not found any band at desired size. I have isolated plasmid from positive colonies but have not found any shift in the size from only plasmid (used for backbone) in 1 percent agarose gel. The recombinant plasmid should be 1.5 kb more than the original plasmid. Why am I facing such kind of problem? can anybody help me with their suggestions?
How large is the plasmid? When running undigested plasmid it will run differently if it is supercoiled or not. You can try linearizing the plasmid or even better, digest with an enzyme that cuts 4-5 times and will give a different pattern with or without insert.
It is possible you don't get product from the replica plate because you took too much bacteria, make sure to only gently touch with the tip.
The original plasmid is 4733 bp long and the plasmid along with the insert is 6248 bp long.
That's a large enough difference that you should see. Another option is that you had an incomplete reaction so you don't get the full plasmid. There is some DNA on the original plate that give you the colony PCR band but once you streak the "positive" colonies on a new plate you no longer see it since it is not really expressed in the bacteria.
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