How to induce self-ligation of a vector? Self-Circularization? - (Jun/03/2008 )
Hey,
since 3 month I am working on this problem:
I have a vector and I wanna decrease the size to 3,5 kBp. So I performed a PCR of the part that is assumed to religate. I added a XhoI restrictionsite to each end of the PCR-product (and also have 4 nucleotids overhang). Afterwards I clean the PCR and digest with XhoI. Next I employ gelelution and try to self-ligate my PCR-Product. Unfortunately the yield is extremely low, I see ~80% of linear fragment, 5% circular vector and around 15% dimer/trimer... products.
My ligation is set up due to the fermentas protocol for self-ligation:
--> 50ng DNA + 5u Ligase + Buffer; fill up to 50µl
For a prospective transfection I need around 2µg of my little vector, so the ligation must be in a extreme huge volume and the I have to dialyse and speed vacc for long. This is not a nice procedure.
I would be really lucky if you have some suggestions/experiences/ideas how to improve this...
Greetings right from the lab.
chris
I used a pretty similar procedure recently and here's what I did differently to your protocol.
I ordered phosphorylated primers (for subsequent blunt end ligation, you can also buy regular primers and use T4 kinase afterwards) and they were HPLC purified (tried before without this cosly purification and some primers were truncated resulting in "wrong plasmids").
I did PCR (with a proofreading enzyme), then digested with DpnI (idea behind this step is similar to why it's done for site directed mutagenesis using quickchange protocol from Stratagene). Purified (qiagen PCR clean up, other methods will do) and then used for "blunt end" ligation and transformed e. coli Dh5alpha. Screened colony's and worked perfect. (did this for 4 different constructs).
What features are present in the vector? Is there no ampicillin resistance gene or origin of replication?
Hey, unfortunately there´s no bacterial backbone (no resistance gene, no Ori), just a CMV-Promoter, eGFP and a S/MAR sequence (for docking onto the nuclear matrix).
Hey, unfortunately there´s no bacterial backbone (no resistance gene, no Ori), just a CMV-Promoter, eGFP and a S/MAR sequence (for docking onto the nuclear matrix).
U want to circularize somehting without resistance gene and without ORI? !
can't possibly get it back into E.coli for sure assuming that the next step is putting it back into E.coli.
won't replicate and no way of selecting . sounds like a gone case.
U want to circularize somehting without resistance gene and without ORI? !
can't possibly get it back into E.coli for sure assuming that the next step is putting it back into E.coli.
won't replicate and no way of selecting . sounds like a gone case.
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Hey,
I know this is a tough job, but my chef wants me to produce this minicircle. Indeed there is no Ori or resistance gene (just an eGFP), so we cannot amplifiy in e.Coli. His idea was to transfect fibroblasts and select them by eGFP. But for transfection I need a small amount of this minicircle. In the moment my yield is too low and every step (Digestion, ligation, dialyse, speed vacc, gyrase, exo III with the change of buffer,...) means such a big loss of DNA. I really don´t like this project either, .... :-(
Can I ask what the goal of your project is?
I did not think you could transfect cells using a ligation mix, but I have no experience with fibroblasts.
u can keep ligation mixture in 4 C at thermal cycler for overnight, it can increase the ligation efficiency
I did not think you could transfect cells using a ligation mix, but I have no experience with fibroblasts.
We want to determine the rate of establishment of this minicircle as a episomal stable vector.
I transfected once with my ligation mix and suprisingly got green cells. But I was not sure if maybe some of my template (it´s a vector 5kb, from which I perform the PCR and religate the PCR-product) left in the mix, so maybe the green cells are due to this origin vector. I want to employ a southern...
Sounds like an interesting experiment. I guess you are trying to identify mammalian sequences that function as origins, otherwise why would you remove the bacterial sequences and why would you need circular DNA. I'll assume, whatever the experiment is, that it is well thought out. Here is what I would suggest, if using phosphorylated oligos does not give you the improvement you need.
Reconstruct your vector such that there are gamma delta resolvase sites flanking your region of interest. Treat with with gamma delta resolvase in vitro. This will give you singly linked catenanes. Linearize the circular molecule that you don't want, then gel purify your circular plasmid, or transfect the mixture. Check out Science. 1992 May 29;256(5061):1298-303 and papers by NDF Grindley for more details. The resolvase is very efficient, so it may be worth the trouble. You will have to get the enzyme from a lab that studies it, I doubt it is sold.
-d