Hi all,

I am curious whether it is possible to directly perform a PCR on your phage display library containing the phagemid. I attempted this and encountered an unexpected result. The primers target the VHH gene, including the tags, promoter, and RBS, so the expected amplicon size should be 642 bp. However, I observed a band around half the size of the insert (see image). I then tried PCR with amplified phages from a successful biopanning campaign (three rounds of biopanning) and found that in round 1, both the expected and a smaller sized band appeared. In round 2, only the small band was present, and in round 3, only the expected size band appeared. Interestingly, the size of the small band does not match the combined sequence of the tags, promoter, and RBS (which is 258 bp). From sequencing the library, we found that only about 5% of the sequence corresponds to the empty vector, so I would not expect the small band to be caused by that.

1) Should i be aware of something when conducting PCR directly on a phage pool (1µl of ~10^13 pfu/ml)

2) Does phages/bacteria cut out the gene of interest in such high amounts?

3) How come 2nd round lack the bigger band and then all of a sudden in third round we only observe the big band?

I hope you can help me answer these questions. Thanks a lot in advance!