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help....hi,
i`m new to cloning, but i`ve read a lot about cloning from articles and textbook.
recently, i tried to clone a ~2kb insert into ~4.5kb pBudce4.1 from invitrogen.
the insert was amplified using primers with at least 6 nucleotides away
from the cutting site. i got a clear band at ~2kb from PCR using fermentas PhusionTaq.
then, the insert was eluted fromn gel and purified DNA conc was ok (~80ng/ul).
i used NheI and NotI to digest the eluted PCR product and i got clear band at ~2kb after gel check.
i also digested the pBudce4.1 using NheI and NotI, total volume 50ul and then eluted
the 3.5kb fragment. Gel check also showed a clear band at 3.5kb.
i tried to ligate this 3.5kb vector with the ~2.0kb insert. the molar ratio
i tried were 1:1, 1:3 and 1:5 using T4 DNA ligase (have tried both 16`c and rt O/N ).
plasmids were transformed into DH5a but there was no growth.
how can this be? my senior said that maybe the insert was not completely cut.
what can i do to improve the cutting?
tomorrow i will transform uncut plasmid to check the DH5a competency,
if the competency is ok, is there anything i could do to optimize the ligation condition?
before this, i have tried cloning into TOPO blunt end with the PCR products,
but i got no growth twice... pls help...
thank u in advance,
TY
