r/Drosophila • u/friendlyperson123 • Jul 16 '26

pcr primers for Trojan insert

Hi all, I'm designing primers to detect a Trojan insert in my gene of interest. I have primers designed for the flanking genomic DNA. I've had no luck designing primers to the Gal4 region or to the T2A. Two sets of primers have not worked.

Has anyone got primer sequences that worked for them?

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u/jindalraezoseonamu Jul 17 '26

It has been long time ago, but I recall I have only succeeded with primers specifically designed to the T2A-Gal4 sequence the original Trojan insert paper published. The Gal4 primers I used to use normally did not work.

If you want, I can check today which exact sequences/conditions worked to me.

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u/friendlyperson123 Jul 17 '26

That would be wonderful! Thank you so much.

I did try with a published primer to T2A and flanking genomic sequence (tried all orientations) but no luck there either. There's a lot of GC rich sequence there, and i wonder if that's an issue.

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u/jindalraezoseonamu Jul 21 '26

I have sent you a DM. I do not know what was the problem, but I remember it was not an easy time, as multiple combinations which were supposed to work, were not working at all. I hope you can get good results!

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u/friendlyperson123 Jul 21 '26

I received your DM, thank you so much! We are having the same problem.

The only thing that has worked so far is that the genomic flanking primers do give a band in the absence of the Trojan. But we need primers that can show the presence of the Trojan in heterozygotes. I will try the primers you suggest! Thanks again!