After transfection with the plasmid ( linearized ) and subcloning of the cell lines, RNA was extracted from the cell and then reverse transcripted to cDNA. When PCR reactions were run to verify the existence of the target gene on the plasmid, the bands of the cDNA were always not the same as that of the plasmid using as the templates. Can anyone tell me why and how to improve it?
Hi Anita:
Can you exclude that you visualize the endogenous gene? Otherwise it ii hard to explain. I would recommend to sequence the product.
Best, Uwe
Did the plasmid have cDNA or gDNA cloned in?
Did you use the same primers for all of the PCR reactions?
Did your cloned insert include an intron?
Did you sequence the unexpected product?
We're going to need to know more information to give better help.
Hello,
I definitely agree with Uwe. Sequencing is the best way to figure out what's happened. Otherwize it is really hard to explain as we don't know your plasmid or insert sequences... For example, it might be due to a splicing process.
Good Luck
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