My vector size is 5.1kb and insert size is 4kb. Initially, I performed digestion of the insert from a different plasmid and the vector using KpnI-NotI. I checked them on the gel, excise and extract the band from the gel. I aimed ligation 1:3 and 1:5 at 4C and 16C overnight and RT 1hr ligation. I used fresh enzyme and buffer. I performed transformation in Stellar cells. However, I got very few transformed colonies on plates where ligation was performed 1:3 at 16C ON. Recovery of Stellar cells was great. Unfortunately, the transformed colonies do not contain the insert. They carried the empty vectors. Any thoughts?
Hello Abdullah,
It's better to check every step.
First, did you use the same buffer for your double digestion? because KpnI-NotI don't have a common buffer for 100% efficiency.
The linearized vectors are flexible and tend to self-ligate, which may cause high background and colonies without the insert.
It's also possible that you have incomplete linearization of the vector, try cutting it again and gel purify.
If your cloning efficiency is low, you may obtain better results if you dilute the reaction.
I don't know much about Stellar cells, but for me, I use Competent E. coli DH5 α, I usually pre-culture at 37C for around 1~3 hours, which is enough to see clear turbidity (cell density of cells).
My suggestion is to experiment with different ligation ratios, beginning with 1:1, especially since you have a large insert relative to the vector size.
Well, the quick answer is the insert didn't get successfully ligated in, as you already know. Ok, now for some thoughts as to what might be going on and what could help.
Your insert and vector are nearly the same size, might be worth trying a 1:1 molar ratio. Try for about 50 ng of vector per 3 Kb of backbone size per ligation.
Did you run any ligation controls? If you want to know how much background (uncut or cut then self-ligated) you can add in two reactions. First one is cut vector (no insert) with ligase and ligase buffer, that lets you know how much vector was uncut or stuck back to itself. Second one is cut vector (no insert) and no ligase but with ligase buffer (just to keep conditions the same), that lets you know how much vector was uncut. Transform these control reactions along with your actual ligation. Ideally, you get 0 to few colonies on the control plates, and more on the real ligation plate. In reality, there are usually some colonies on all the plates.
Do you use two different restriction enzymes for the two insert ends? Are both sticky? It can be a challenge if one or more leaves blunt ends.
Another issue can be if your donor plasmid and destination plasmid have the same selection marker. I had this happen before and I would get back the original insert vector. I added in a third enzyme to cut in the vector backbone (but not in the insert). That did work.
Hope this all helps!
When you're doing double digest, especially, if the enzymes do not work at 100%, include single digest controls to see, whether both of your enzymes work sufficiently.
Additionally, you can include a killer cut. If there is any restriction site between the two you are using, you can include a little of this enzyme at the end of the restriction to digest any remaining plasmid.
Amy (and I elsewhere) wrote you already about the controls for the ligation. This should answer your question where is the problem.
Also, make sure your ligase and buffer are fresh, the buffer can expire or degrade, especially if it has gone through multiple cycles of freezing and thawing.
Tomáš Hluska So the steps are - digestion, band excise from gel, ligation and transformation. After double digestion, I saw expected sized 2 bands on the gel. This means, digestion is not the issue. I cut the band that I want from the gel. After extraction from gel, 260/280 ratio of extracted DNA was 1.5. I used same competent cells to transform different plasmids. It worked fine. I guess, ligation is the issue.
What is the size of your plasmid and of the part you cut out? I bet the difference is at least 1:300 (10 nt from 3000 bp plasmid). You have no chance to distinguish, whether your plasmid is double- or single-digested, if you perform only double digest.
Anyway, use proper controls for the ligation and you will see immediately, where the problem is.
Tomáš Hluska Size of the plasmid 6.8kb and that of desired fragment 4kb. After double digestion, I found 4kb and 2.8kb band. I extracted 4kb band from the gel and tried to ligate with vector of 5.1kb.
Abdullah Al Tarique You mean the plasmid from which you take your insert, right? But I mean the plasmid into which you clone it. How big is it?
Anyway, the best thing to do is to include all possible controls for the ligation.
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