I am trying to clone 2 fragments of 1.6 and 1.1kbp into pGL3 basic vector (4.8kbp) using In-fusion HD cloning kit (Clontech).
I added totally 90ng of 1.6kbp fragment and 70ng of 1.1kbp fragment with 150ng of linear vector into 10ul reaction volume, incubate at 50oC for 15min and then transformed into Top10 competent cells. However, I could not obtain any colony on the agar plate.
I used to clone with 3.2kbp fragment and it worked well.
Do you have any experience or suggestion about this work?
Thank you very much for your help.
Re-check the overhangs for homology, there might be a problem. Happend to me as well. If the kit is old, you also might want to check whether the enzym is still working. Cheers
Thanks Oliver,
I check the homologous sequences of the fragments and vector, they are completely ok.
Have you every tried clone 2 fragments?
Indeed I did. The kit worked for me to clone up to three fragments in one go with inserts ranging from 100bp to 2-3kbp. The only problem I faced so far are mistakes in the pimer design. However, sometimes I get only 3-4 colonies after transformation. These colonies are usually 100% positive. As far as I understood, you do not get any. For me it seems that either the enzym is dead (just check with the positive control included in the kit) or that the bacteria are the problem. Have you tried different bacterias? And just to make sure, did you clean your DNA with a kit/gel purified it after the PCR? Sometimes I used the PCR-reaction mix without cleaning the DNA, however the recombination efficiency decreased resulting in very few colonies. Maybe you want to change something there. Else I always used the manufacturers protocoll.
Hi Oliver,
Thanks so much for your helpful reply,
Could you please tell me in detail the concentration of each fragment and vector used for in-fusion cloning?
I am very glad to find someone out there using this method...
Hope you could help me out of this tricky problem...
Thanks again!
Hey Leo,
I wanted to share this link in my previous post: http://bioinfo.clontech.com/infusion/molarRatio.do
You will find a calculator that will give back the ng of DNA you will have to put in your reaction. I always followed the instructions you will find there.
Good luck with your cloning.
Cheers
Hi Le Duy,
Did you overcome your problem? I'm getting the same one:(. I'm cloning 2 fragments(1.5kbp and 420bp) into YEplac195 vector (~5.2kbp) using In-fusion HD cloning kit (Clontech) but i failed even i tried changing DNA fragment concentration.
I'm very happy if you coul share me some tips in this case.
Thank you so much!
I am sorry but I gave up the experiment.
I only succeeded cloning with 1 fragment.
If possible, you can contact with Clontech to ask for assistant.
Good luck!
I have done more than 20 plasmid constructions by using infusion cloning kit. Usually my insert is around 1kb and my vector is ~10kb. I recommend EB 10-beta Competent for big vector (~10kbp). Recently, I just finished cloning 2 fragments of 1kb and 508bp into pBlue vector (4.75kbp) using In-fusion HD cloning kit (Clontech).
Hi everyone, I have succeeded in constructing the chimera with 2 and 3 fragments in pMSCV neo vector but sometimes I also obtain many colonies which don't contain the insert. That's a big problem so I have to run colony PCR to check that plasmid is correct or not. I suggests to prepare the linearized vector by restriction enzymes with longer incubation (over night is better) then check the background by transformation step to assure that the vector is completely digested. Protocol that I used is following:
purified pcr fragments: 80ng/ul
linearized vector: 20ng/ul
5x infusion hd enzyme premix: 2ul
D.W: up to 10ul
In my case, after amplifying the fragments by PCR, I always purify PCR products by QIAEX II gel extraction kit.
Hope this helps.
Cheers.
I too am using this method for a very large plamid. I have a vector (Backbone) for ~4kb, and 3 inserts (each aprox 4kb) as well.
I have suceessfully PCRd all of them - Including the vecotr (not by restriction digest as some do because there are no sites to cut where I want/need).
After successful PCR - I had to troubleshoot a few times to gel extract bands that had high enough DNA concentration to properly use in the InFusion reaction. (between 30ng- 60ng concentration). I think the higher, better quality DNA after gel extraction is a big factor in a succesful InFusion reaction. Also, using their molarity calculatory on Clontech website is really helpful and I am to use those ratios as well.
A lab next to mine routinely does InFusion with lots of success so I am trying to learn from they. They commonly only get a few 10's of colonies after transformation.
I recently had a successful transformation of my 3 insert/1 vector reaction and will be doing min preps/sequencing soon.
A somewhat successful previous attempt yeilded 4 colonies and I mini preped them and sent for sequencing where the sequencing yelided the restult of the Vector only- and the part of the vector that I didn't PCR?!
This was really frustrating and I'm not sure why so I will be taking a few steps back.
Any suggestions and updates going forward from the community working on Infusion is always welcome!
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