Improved colony PCR to screen for and identify recombinant clones carrying partial segments coding for core proteins of the duck hepatitis B virus

Objective To verify the efficiency of improved colony PCR.Methods Partial overlapping fragments of core proteins of duck hepatitis B virus(DHBV) and segments coding for DNA polymerase(DHBc-dp) were amplified by polymerase chain reaction(PCR) and the products were purified and cloned into the T vector pMD 18-T.DH5α component cells transformed with a ligation solution were plated onto LB plates with twice as much Amp.Plates marked as the parent sample(PS) were incubated at 37 ℃ for less than 12 h and then left at room temperature for no more than 8h.Sterilized tips were used to ascetically collect predominant resistant colonies.Bacteria were then amplified with PCR solutions and identified while remaining bacteria were left at room temperature to undergo secondary growth.Remaining PS PCR products were transformed into DH5α component cells that were plated on LB plates;these cells were designated filial generation 1(F1S),and colony PCR was performed.Samples from the PS and F1S that were highly positive according to PCR were amplified and PCR of the bacterial solution and plasmids was performed.Restriction enzyme digestion and DNA sequencing were also performed.Results After colony collection,colonies had extensive secondary growth.PCR of predominant colonies provided equivalent results and even provided better reactivity and consistency than conventional PCR.Recombinant plasmids carrying the insert in question were retained the same structure during colony PCR and were faithfully reproduced and amplified in the F1S.Conclusion Improved colony PCR retained the same specificity and efficiency with regard to recombinant clones as colony PCR had while effectively preventing the loss of positive transformants.The improved method can effectively inhibit the growth of satellite colonies despite the greater growth of predominant resistant colonies.