The protein was expressed using the insect cell system, and the recombinant plasmid was successfully constructed as verified by sequencing, and the recombinant plasmid was transformed into DH10 receptor cells for screening of blue and white spots, and the selection of white spots was verified by PCR reaction, and agarose electrophoresis showed that all of them were negative.
I'm a bit confused. Could you draw a step by step diagram of your cloning and screening process?
Hello Alexandra Johnson ,
The target gene was cloned into pFastBac series vector to produce recombinant plasmid, and the insertion sequence was verified by sequencing.
The pFastBac recombinant plasmid was transformed into DH10Bac Escherichia coli and cultured at 37℃ for 48 hours. Recombinant strains were screened by blue-white spot after transformation, and the recombinant strains showed white spot due to the damage of LacZ gene caused by transposition. The LB agarose plate used for blue-white spot screening contained 50μg/ml kanamycin, 7μg/ml gentamicin, 10μg/ml tetracycline, 40μg/ml X-gal and 40μg/ml IPTG.
White spots were selected and the colonies were further verified by PCR. The PCR reaction uses pUC/M13 forward primer (5'-CCCAGTCACGACGTTGTAAAACG-3'). And pUC/M13 reverse primer (5'-AGCGGATAACAATTTCACACAGG-3').
By negative PCR reaction, do you mean you're getting a wild-type sized band or no band at all?
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