Hi,
i faced some of southern blot problem.
my chlamydomonas transformants vector is pChlamy RNA3. and vector has APH8(paromonycin) region. So i designed APH8 probe.
probe size is 350bp.
After southern blot detection step, my wild-type also had band with transformants.
But wild-type didn't have paromomycin region.
i don't understand that.
the other problem is restriction enzyme.
i used Bgl2 and Pst1. and treated 37°C 16h in pcr machine.
But after southern blot detection step, every band location was same.
my southern blot kit is amersham gene images alkphos direct labelling and detection system. and membrane is hybond-N+, detection kit is CDP-star.
thanks for your answer.
I have only an answer for your problem. I think that your wild type is contaminated with your probe or vector. You can follow two ways. The first one is: throw anything (buffers, loading buffers, kits and DNA samples, anything) in the waste basket, wash your pipets, change your filtering tips and re-extract your samples. The second one: make a southern blot with the different components used, including one well with only loading buffer.
You forgot to say one thing. The lengths of Bgl2 and Pst1 fragment(s) correspond to the vector map?
Good luck
Andrea Rando
You take older unused or preserved stock of chlamydomonas which was never used after the transformation experiments to rule out the contamination.
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