I did a ligation between a 9kb vector and a library-pool of inserts 97 base pairs long. I tried three different ratio: 2:1, 1:1 , 1:2 and I need to avoid insert concatamers.
After transformation, I had many colonies for all three ligations and none in the vector-only control.
Then I checked the correct size of my insert by colony-pcr followed by tapestation analysis. The expect amplicon is 285 bp long and vector without insert should be 221 bp long.
I attach here a picture: from left to right , 3 colonies 2:1, 3 colonies 1:1, 3 colonies 1:2 and vector only control. As you can see I have a nice correct size bands at the expect size (around 285 bp) . However, what are others heavier bands that are shown in several samples? Are they PCR artifacts( I used 35 cycles) or are they coming from 97 bp insert concatamers being linked to the vector and transformed as well with the correct one?
Thank you so much for your help
Without further analysis I don't think anyone can tell you what those other bands are beyond taking a guess.
Do your insert have terminal phosphates on them? If they don't then there should not be a problem with multiple inserts. If they do then it is quite possible that you have a small population of multiple inserts. But if you assume that 10% of your population is concatameric, is that really a problem or can you screen them out at a subsequent step. Sometimes we spend more time trying to solve a problem that we can ignore. But it depends on the specifics of what you are doing.
Thank you for your answer.
Insert were just digested with two restriction enzyme so they should have phosphates.
The whole DNA of the plasmid population will be extracted and then a second restriction digestion and ligation with another insert will be performed. This second restriction and ligation site is within my 97 bp first insert.
I don't know what happens if I have also concatamers of inserts there...
I wonder how is it possible that I have a colony with correct insert and some concatamers: this would imply that multiple different plasmids enter the same cell but I think the chance of this event should be extremely low....isn't that the case?
If one of the restriction sites for the second cloning is within the insert then there should not be a problem. Assuming the vector was completely digested, then your multiple inserts should only be in multiples of 3 (F, R, F) in order to have the proper restriction site orientation. In your second cloning the restriction digest will generate a vector that is identical between the monomer insert and the trimer (or higher) insert.
1 and 2 represent the two ends of the insert and r is the internal restriction site:
1----r-2 2-r----1 1----r-2
It would only be an issue if some of the vector was only cut by one enzyme so that you have inserts that are oriented F-R in order to have the same restriction site at both ends.
Thank you,
I also thought about this and maybe I will proceed after Sanger sequencing some colonies just to confirm.
If some vector is cut just by a single restriction enzyme, then maybe some of them would reanneal and I would see some colonies in my no-insert control. I see zero luckily.
I just wonder if those bands could just be PCR aspecific amplification from colony pcr coming from too many cicles
Also those bands are different for every single colonies and doesn't look like multiple of 97...it's hard to understand them for me.
Also one bacteria statistically should receive just a single plasmid? If correct size plus concatamers, this would imply several different plasmids enter a single bacteria. Chance is very very low or maybe I am wrong.
- Badges
- Science topic
- Similar topics
- Phytochemistry
- Extracts