How to generate a UAS line | Need Guidance

[‍무하마드아티프(대학원생-바이오발효융합전공)]

Dear Community Members,

I am a new fly research student. I need to generate some UAS lines for the Ir receptor gene in drosophila for my project. For the past 2 months, I have been trying to do the cloning steps but I am not even getting the sequencing correct. It's impossible to share the whole protocol here but I will be happy if somebody can guide me or refer me to some materials or protocols so I can follow them to generate the UAS lines.

Additionally, since I am new here it would be good if you could ask me the questions and I will answer you accordingly.

Just shortly, I will put here the protocol I am using

mRNA extraction > PCR amplification using the primers that contain the restriction enzyme sequence > Gel extraction using bead method > Ligation using T-blunt vector > Transformation using Dh-alpha competent cells > sub-culturing > mini-prep for T-vector DNA > Restriction digestion > then sending the Tvector DNA sample for the sequencing using the M13 primers.

Kindly help me in this regard. I would be very grateful for any support.

Thank you for guidance in advance.

Yours Sincerely,

Muhammad Atif