METHOD FOR PREPARING HIGH-THROUGHPUT SEQUENCEABLE DNA FROM INDIVIDUAL PLAQUES OF PHAGES PRESENTING PEPTIDES
The present invention relates to a process for isolating DNA from individual plaques of peptide-presenting bacteriophages in a high-throughput capacity PCR, wherein the PCR products obtained are sequenceable and a specimen of each phage studied is retained in a replicable state. The PCR is successful despite the presence of inhibitory constituents from the growth medium or the host bacteria.
1 . Process for the high-throughput capacity preparation of sequenceable DNA from individual plaques of peptide-presenting phages with the simultaneous retention of infectious phages, comprising at least the following steps:
A thinning out peptide-presenting phages from a phage population on a growth medium containing host bacteria,
B amplifying the thinned out phages by means of incubation,
C picking the phages from the growth medium and suspending them in a medium,
D lysing a portion of the suspended phages from step C and also using this DNA-containing lysate as a template in a polymerase chain reaction (PCR).
2 . Process according to claim 1 , wherein, in step C, phage plague is picked from the growth medium with a sterile tool and, by this means, the plague is pricked out from the agar plate to yield an agar plug and subsequently the agar plug is transferred into the medium.
3 . Process according to claim 2 , wherein the suspending in step C is carried out with an input of energy.
4 . Process according to claim 3 , wherein the volume of the medium is at least twice that of the agar plug and the agar plug is completely covered in the vessel used for the suspending.
5 . Process according to claim 4 , wherein the lysis in step D is carried out within 8 hours at most subsequent to step C.
6 . Process according to claim 5 , wherein the lysis in step D is carried out by adding an aqueous lysis buffer which comprises at least one buffer substance, one surfactant and one complexing agent.
7 . Process according to claim 1 , wherein the lysis in step D comprises heating to a temperature between 80° C. and 98° C.
8 . Process according to claim 1 , wherein, in step D, an amount of between 1 and 30% by weight of DMSO is added to the PCR.
9 . A selection process for the binding phages from a phage display library, said selection process comprising a process according to claim 1 .