I tried partial digestion for the genomic DNA (bacteria) by Sau3AI, I digested the DNA in a very short time (~ 30 mins), then the reaction generated a smear band in gel, I cutted the band (1-2kb, and no more longer DNA segments in gel) to ligated to pUC19 (BamHI digested, CIAP treated), I did this very successfully, I generated the plasmid library and examinated them by sequencing.
However, the DNA length is too short for me. So that, I try to shorten the digestion time(~20 min ) and the lowered concentration of the SauAI, and recoveried the DNA band ( 3-6kb) from gel. This trail is totally failed. No single combinant found.
So my question is , why I cannot do the ligation like I did before.
The efficiency for pUC19 to accept larger DNA fragment has become ineficient.
The efficiency may go down but 3-6 kb are not a problem for pUC19. My recommendation is very simple: Do it once again.
After transformation, do not directly plate onto LB agar (plus antibiotic). Instead, transfer to LB broth (plus antibiotic). If your culture turns turbid...you will mostly find plasmid with inserts....:-).
Then you plate on LB agar (plus antibiotic).
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