I did a Gibson assembly reaction and consequently, I did the transformation with my construct to E. coli DH5alpha and I plated the bacteria into 20 µg/mL Kanamycin agar plates. The first time the colony PCR result was successful, however, last week I did another colony PCR with the same colonies that I chose (which I had frozen to make the backup), and this shows primers dimers. Idk what's going on, do you have some ideas about it? My positive control was effective and showed the respective band in the gel.
Cloning voodoo, I'd just remake the primer working stock and try the PCR again. If possible, make a miniprep using the positive colonies (I mix 20uL water with the colony and take 5uL for a colony PCR) and do the PCR on purified plasmid DNA, colony PCRs can be very messy.
Cole Peters Thank you so much! But I purified my plasmid from bacteria before each colony PCR and idk why I can't obtain the same results as the first time, I thought that maybe my bacteria lost the plasmid but I think this is impossible because I maintained the transformants colonies froze at -80ºC with 20% Glycerol and LB + Kanamycin.
If you still get the right size bend (showing that your plasmid is in) I would not worry about primer dimers
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