I am performing phage display and I occasionally find that some of my ELISA positive clones when isolated and sequenced give me phage sequence and not the phagemid. Any experience with this? Any insider knowledge on why this happens?
It sounds like you are enriching either plastic binders. Phage on its own at high amounts can stick non specifically to the solid phase. Was the background low or high in the immunoassay? Perhaps try more stringent washing. Pls Check if you are plating the clones out on the correct antibiotic agar.
Theam Soon Lim , I tested clones on plates with antigen and also on plates with only the blocking protein. Clones which were only positive on the antigen coated plate and negative on the blocking control plate were moved forward. I am restreaking the positive clones on both carb (the phagemid marker) and kan (the M13KO7 marker) plates to see if clones are only positive on carb. I will update this feed as I get additional information.
Follow up on the originally posted question.
I restreaked the clones onto both carbenicillin and kanamycin plates. There was only growth on the carb. plates. I selected new colonies and performed minipreps. The yield of the minipreps was almost 10-fold higher compared to the original preps. Each clone gave beautiful and unique sequences for VHH.
In addition, I downloaded the sequence for M13KO7 and found that the phagemid primer that I used is also in the helper phage genome. My conclusion is that the original clones must have had sufficient helper phage contamination and this was subsequently purified during the miniprep. Can anyone infer a better explanation?
I will add restreaking of any ELISA positive clones for future screening to reduce the chance of repeating this event.
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