GFP band size1kb
IPC band size 324 kb
PCR is already standardize...positive control is giving band for both IPC and the transgene(GFP)
Hi Ipsita,
Have you first confirmed GFP-tagged protein is functional or not ? Alternatively, you can use the RE and set up digestion for a couple of hours and run the samples on an agarose gel. Correct insertion would give you two bands, by which you can confirm the right insertion.
Sounds like you are doing genetic transformation to produce transgenic lines.
I guess that your 'positive control' for PCR was the plasmid with GFP gene, which was used for transformation. What 'internal control' gene did you use for PCR? EF1α? It will not tell you that whether your lines are transgenic even the 'Internal control' was amplified (your attached picture). But, it tells you that the isolated gDNA is suitable for PCR, without such as 'PCR inhibitors' interference. Your lines may not be transgenic.
Have you tried to amplify selectable marker gene, such as Kanamycin-resistant gene, nptII)?
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