I have been trying cut out a 3.3kB vector from a plasmid vector of 19kb and inserting another 3.3kB vector with mutations introduced back into the vector. I have gone through every issue that I can possibly have and have pinpointed ligation as being my issue.
Because even when I just cut the 3.3kb insert out of the plasmid vector with PacI and EcoRV and try to re-ligate it back in, it doesn't work. I have used two different T4 DNA ligase and Takara DNA ligase LONG (which is supposed to be good for long bands) and left the ligation to run for 16degrees overnight. and I seem to get about 50 to 60 colonies after transforming but after purifying the plasmid I have a band at 2kB each time with some higher bands. I tried cutting this purified plasmid with EcoRV or PacI, all the higher bands seem to go away but I am left with a really bright band around 2kB.
Please help!
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Posted 16 January 2011 - 09:48 AM
amisra2, on 16 January 2011 - 08:52 AM, said:
I have been trying cut out a 3.3kB vector from a plasmid vector of 19kb and inserting another 3.3kB vector with mutations introduced back into the vector. I have gone through every issue that I can possibly have and have pinpointed ligation as being my issue.
Because even when I just cut the 3.3kb insert out of the plasmid vector with PacI and EcoRV and try to re-ligate it back in, it doesn't work. I have used two different T4 DNA ligase and Takara DNA ligase LONG (which is supposed to be good for long bands) and left the ligation to run for 16degrees overnight. and I seem to get about 50 to 60 colonies after transforming but after purifying the plasmid I have a band at 2kB each time with some higher bands. I tried cutting this purified plasmid with EcoRV or PacI, all the higher bands seem to go away but I am left with a really bright band around 2kB.
Please help!
Because even when I just cut the 3.3kb insert out of the plasmid vector with PacI and EcoRV and try to re-ligate it back in, it doesn't work. I have used two different T4 DNA ligase and Takara DNA ligase LONG (which is supposed to be good for long bands) and left the ligation to run for 16degrees overnight. and I seem to get about 50 to 60 colonies after transforming but after purifying the plasmid I have a band at 2kB each time with some higher bands. I tried cutting this purified plasmid with EcoRV or PacI, all the higher bands seem to go away but I am left with a really bright band around 2kB.
Please help!
I've probably got less molecular cloning experience than you have, but, with that caveat out the way, you could try ligation for a few days at 4 degrees Celcius? That's what Promega have recommneded for people having problems.
Also, are you sure that your enzymes aren't cutting somewhere inside your insert? That would explain the 2 KB fragment.
