Hi, hope you're doing well in your research.
I have been doing restriction enzyme digestion and ligation to construct plasmids for two weeks and I got very unexpected self-ligated plasmids. The reasons why I call it unexpected are :
- I used two different enzymes (Bam HI+Not I) to cut my vector (from plasmid A) and the insert (720bp, from EGFP-N1).
- Agarose gel showed my insert was successfully cut from EGFP-N1 as the band around 700bp was very clear.
- Agarose gel showed my backbone was successfully cut. Plasmid A was cut with both enzymes and gave me the backbone and a 1000bp fragment.
- I ligated the insert and the backbone with different molar ratios (1:3, 1:5, 1:7, and 50ng backbone was used). I used ligation high from Toyobo to ligate and I ligated for 2 hours at 16 degrees.
- Only the one with 1:7 gave me a few colonies and I sequenced all of those plasmids.
- I got self-ligated EGFP-N1.
I don't know why I got self-ligated EGFP-N1, as I only cut the 720bp EGFP and the backbone of plasmid A. I'm sure that I didn't cut the EGFP backbone by mistake because I verified the insert and the backbone again using the agarose gel.
I wonder if there is an explanation for this, also I would like to hear advice from you about improving ligation experiments.
Thank you so much for your help.
Well, if both plasmid A and your EGFP-N1 vector have the same selectable marker you can get backbone carryover. You could try adding another restriction enzyme to your EGFP vector digestion (one that will destroy that backbone and not cut your EGFP), that will help to reduce vector carryover.
For faster ligations I like to use NEB quick ligase, it only needs 5 min at room temperature. It's good to see you are using different ratios of vector:insert. As a note, the 50 ng of backbone is for every 3 KB in size. So, if your vector is larger you'll need more of it (example: use 100 ng for a 6 KB vector).
Hope this helps!
Amy Klocko Thank you for your advice, Amy. I'll try to eliminate the EGFP backbones by adding a third enzyme and increase the amount of my vector.
In addition to Amy Klocko answer, another thing to remember is that when double digesting your backbone plasmid, some tiny fraction may have only been digested once. These might not even be visible on a gel. But of course they will recircularize with high efficiency and give you background vector transformants. Although I would have expected to see some in all of the ligation reactions that have vector, so that is a bit surprising.
It may be that the main problem is very inefficient transformation, I would have expected you get some colonies from all of the different ratios. You might wish to do a control transformation and be sure your cells are really as good as they should be.
Thank you Michael. Michael J. Benedik My transformation efficiency is always low (usually no colonies) when I'm doing ligation or infusion cloning while transforming competent cells with intact plasmids has never been a problem.
Previously I thought there were no colonies because the vector: insert ratio was not suitable. But it seems like those ratios should also give me some colonies, instead of 0 colonies.
I'll just do a control experiment in case anything wrong with the plates or reagents.
If you transform with an uncut control plasmid you should get tens of thousands of colonies. I've seen lots of people get a few hundred colonies with a control plasmid and think their competent cells are fine, but in fact are 2-3 orders of magnitude low. You should nearly always get some colonies with a transformation (assuming you actually still have DNA) if your cells are good.
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