Smallest possible insert size for Ligation reaction - (Jun/14/2013 )
This would be my very first post on this nice forum. I hope for some nice and helpfull suggestions and experiences. I was wondering if somebody could share his/ her experience with me about, what can be the smallest possible size of an insert which can be succesfully ligated into a very huge plasmid (around 17 kbp), and is there is any scale for the size of the insert and the size of plasmid for a better ligation reaction?
Thanks all in advance.. looking forward to hear from you.
In theory any size is possible, in practice anything under about 10 bp will be very tricky , just due to the physical size of the ligase and steric hindrance. As to the size ratio, I find that plasmids that are about 5x as large as the insert work well, but this may just be because I am using a plasmid that is "easy" to clone into.
The insert has to be large enough to not denature, since only dsDNA will ligate. From a practical standpoint, a dsDNA region of about 12-15 bp is probably sufficient. Remember when attempting this ligation that you need very very little of the insert to reach equimolar amounts -- you'll probably dilute your insert oligos by 1000x or more.