Hi all
I ligated my small insert (37bp) into 3.5kb plasmid, I got colonies but when I check the sequence of my insert, I found more than one insert ligated as a repeats, some clone got three inserts toghter while some clone got 5 inserts, it seems that the insert ligated to itself.
Anybody got this problem? and how we can sort it out?
thanks for your help.
that sometimes happens when you have smaller adapters (or larger ones for that matter). Reduce your ligase concentration and ligation time.
Dear Hadi,
This is quite common. I wanted to first ask if you designed your insert sequence to be phosphorylated? If so, this often results in the multimer insertions you are seeing. One thing I found to stop this is by ordering primers and/or DNA blocks to not have any modifications; once the two DNA pieces find each other, the cell will do the rest. Hope this is helpful and I would be happy to talk more/share my protocols if you would like to discuss more?
Without changing your design (using non-compatible ends), it basically boils down to adjusting your ligation parameters. As previously mentioned, use less ligase and shorten the ligation time (15 to 30min max with kits like Takara), but also reduce the amount of insert and increase the amount of vector you add to the mix. I would also suggest heating up your insert to ~95°C just before adding it to the ligation mixture, and then straight into the16°C water bath.
If the system/kit you use calls for overnight at 16°C, first repeat without changing any concentrations, but then only let it go for ~1hr.
Hello Hadi,
Try reducing the molar ratio of insert to vector, it makes sense that with an abundance of insert molecules they could ligate in odd numbers to give the correct sticky ends for ligation to your vector (making the assumption you are using two different enzymes with different overhangs).
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