I have been trying to clone a 50bp shRNA gene insert into a plasmid 8.8kb long with compatible sticky ends. For some reason the ligation reaction is not effective and so the transformation is not working (I used positive control for transformation also to show it is not my transformation protocol). I used T4 DNA ligase and incubated reaction for 2-3 hours, and also have used Quick ligase for 10 minute (both are from NEB). I decided to run all my sample reactants on a gel to see if there was some sort of contaminant in the samples and I observed an odd band at 1.5kb in each of my ligase samples, 2 of which were brand new (I ran 3 samples of different ligase with milliQ H2O). I know it is not the H2O since i ran that separately beside and there were no bands.
I am just wondering if it is possible that RedSafe can bind to ligase or if there is usually some form of substrate in a ligase sample that may be causing this unusual band.
Ligase samples and unusual bands are highlighted in red boxes. lanes 2, 11, 13
Hmm, that band doesn't quite look like DNA. It seems possible that you are indeed seeing the ligase protein itself. If you want to run one more test, try adding different volumes of the ligase to the gel. If it is indeed a ligase band, the intensity will change.
But, your real concern is why your ligations aren't working. You have a very large size difference between your insert and vector. The standard 3:1 molar ratio for insert:vector might not be ideal. You could try a higher ratio such as 9:1 and a lower 1:1 ratio, sometimes that can help.
As your vector is a bit larger, you may need to use 150 ng (standard is 50 ng per 3 Kb of vector size). As your insert is very small, you will need to dilute it a lot to get to the desired molar ratios.
Hope this helps!
Hi Amy, thanks for these suggestions! Good point with the sizes of the vector and the insert. my current protocol has the molar ratios of inset:vector at about 4.5:1, but I think I will adjust and see if that works. I have noticed also that just transforming full length plasmid works easily when I use about 300-500 ng and produces a good number of colonies with the competent cells I am using. Although, right now my concern is about ligsting enough Dna, so will adjust, thanks for your help! Amy Klocko
Good points from Amy Klocko . I would also recommend a longer ligation incubation time. You can try an overnight ligation between 16°C and 20°C, but temperatures depends on your enzyme, some enzymes works better at 4°C.
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