I have a problem with amplifying the final cloned vector after using gateway cloning (LR clonase). My vector is 5kb and my GOI target is 4kb long. I tried to do a gateway cloning with pDONR221-my GOI and pT3 vector which carries attR1 & 2 flanking regions. The cloning was successful (confirmed by DNA electrophoresis and DNA sequencing). I could extract DNA using mini prep after 12 hours of incubation. The DNA extraction failed for further incubation or midi prep of the bacteria from the same colony, the DNA pellet cannot be eluted. However, the bacteria DH5a continues to grow.
I made sure that the protocol of midi prep didn't have any problem. It seems like the DH5A lost the vector.
Does anyone know if it is related to gateway cloning problem?
My first guess is that this does not look like a midiprep cloning. I would test these two options:
- respread your colony in a fresh plate with the appropriate antibiotics.
- re transform competent bacteria from the DNA you extracted with minipreps
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