I did TA cloning with instaclone PCR cloning kit and I got some weird results.
I picked 12 white colonies, isolated them and cut with a single cutter enzyme BamHI (I cut with BamHI to linearize the plasmids and see the sizes). After restriction enzyme digestion I got the result below.
As it can be seen, I got 2 bands at 3 kb and those are my plasmids that did not take up the insert. I got 4 bands at 4.5 kb and those are most probably my constructs (plasmid=2.9 kb, insert=1.5 kb). But the strange thing that lead me to write here is that I got 6 constructs that is located around 7.5kb.
I am wondering how is that possible to have constructs located at 7,5 kb even my plasmid is 2.9 kb and my insert is 1.5 kb.
Is it somehow possible to have ligation of two or more inserts with a plasmid in TA cloning ?
Note= I purified my insert before ligation process.
Hey Furkan,
Did you run a gel with the PCR you used to generate your insert?
If I remember correctly the TA kit works by ligating DNA fragments with an overhanging A to the vector (PCRs generate an overhanging A), so it could be possible you ligated two fragments together, but I'd be surprised that that happened with 50% efficiency.
Alternatively in your original PCR you had two PCR products that came out in the clean up. One is longer and ligated into the TA vector.
You could always try a few other test cuts or try for a sequencing read across the two insert sizes. I think the TA vector has M13 universal primers flanking the insertion site.
Good luck,
Chris
Dear Chirostopher,
I loaded my PCR to gel and the size was 1.5 kb. As you said my insert has 3'A overhang and my vector has 5'T. In my case, its obvious that more than 2 insert (most probably 3 ) insert ligated with plasmid but I dont understand how is that possible with 3'A of inserts.
Regards,
While in theory you should not be getting double inserts, strange things often happen in cloning experiments. It usually isn't worth worrying about since you frequently will see them. Just select the right clones and move forward.
Thank you @Micheal I will send constructs in the picture below to screening
@Sundar, I am using taq DNA pol for my ligation protocol. As you now taq DNA is 3' A that is why I did not have problem during the generation of constructs as you can see in the picture below. My concern was about the generation of larger constructs than its expected size as you can see in the picture above. But as dear Micheal Benedik said, generation of larger constructs than expected size is not worth worrying.
I will just wait and see the result of secreening.
Regards,
Furkan Oflaz So, what was the answer? Did you have 2 inserts in the vector?
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