I transformed my ligated plasmid onto LB plates with ampicillin. I transferred the colonies to LB broth. After plasmid extraction, I could detect my insert genes with combinations of primers. But the original vector specific primers do not amplify any product.
I have a doubt here.
When transferring colonies to LB broth, if I touch the agar, do the ligated plasmid mix be transferred to the broth? And if yes, will it be extracted along with the plasmid in the elute? Is it possible?
Where exactly are the vector specifc primers? Around the multuple cloning site? What you desribed sound like contamination of PCR product. Can you write down the DNA amounts of your ligation?
My insert gene size is 3185bp. And the vector size is around 6000bp. I have three internal primers to check my insert gene.
The vector specific primer binds 100bp upstream mcs. The vector specific primer and the insert specific primer do not amplify. But insert specific primers amplify the insert genes. Rf cloning was done so I dont suspect vector contamination during restriction digestion
Did you try extracting the plasmid? You can then cut it once with a restriction enzyme and check the size that should be either 6kbp or around 9 kbp.
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