Published: November 22nd, 2019 Last Modified: November 25th, 2019
Manipulating plasmid DNA (which we’ll colloquially call cloning) is one of the hallmarks of modern molecular biology. Yeah yeah, I hear you, gene synthesis is coming to take our jobs away, it will render our cloning skills useless, etc etc. Keep your shirt on! Even if DNA synthesis rates keep dropping often it’s more cost effective AND faster to do some or all of the assembly of the fragments yourself.
Now, we’ve come a long way from when your grandmammy or grandpappy synthesized their own primers and purified restriction enzymes to make the first plasmids. My first forays into modern cloning techniques hopped from ligation independent cloning (LIC) to sequence and ligation independent cloning (SLIC) and finally settling in to Gibson assembly as my method of choice. You have a mastermix, you mix it with the DNA you want to assemble, you transform it, et voila! You (hopefully) have your plasmid.
Problem is, commercial 2X Gibson mix is often between 10-20$ per reaction, and the homemade stuff can only be made at about 3$ per reaction. On top of that, Gibson mix has a notoriously poor shelf life. I have good news folks, you can now make a drop in replacement for Gibson mix at less than a CENT per reaction!
The TEDA protocol, by Yongzhen Xia, Kai Li, Jingjing Li, Tianqi Wang, Lichuan Gu and Luying Xun, was only published in 2018, and it really goes to show that it pays to stay on top of current protocols! It’s a really good read, and they break down the cost of various cloning methods. TEDA cloning comes out on top at less than a cent and I can tell you personally that’s not an exaggeration.
Basically, TEDA cloning does away with all the enzymes in typical Gibson mix and leaves literally only the T5 Exonuclease, here’s the recipe:
5X TEDA Cloning Mix (1 mL)
500 mM Tris–HCl pH 7.5 (0.5 mL of 1M stock)
50 mM MgCl2 (50 uL of 1M stock)
50 mM dithiothreitol (100 uL of 0.5M stock)
0.25 g of PEG 8000
1 μl of 10 U/μl T5 exonuclease (NEB)
Add the liquid ingredients to a 1.5 mL tube, add the PEG, apply some heat to melt it. After everything is dissolved, cool it down on ice and bring it up to 1 mL with dH2O. Add your T5 Exo, mix well, aliqout, and you’re done!
To use, follow typical Gibson assembly rules. The fragments you are assembling need to have at least 20bp of overlap with each other, longer is preferable for >2 or 3 fragment assembly. Gel purified PCR fragments are preferred, and unlike similar mixes like SLICE extract, TEDA will work with PCR generated vectors! Hooray!
Combine your fragments in molar ratios preferred by those knowledgeable in the art, 1:3 vector to insert for 2 fragment assemblies, 1:1:1 or 1:3:3 for multi fragment, you get the drill. You’ll be working in the fmol range, typical assemblies usually have about 200 fmol of fragments total, although you can get away with 50 fmol (pretty grim) or higher like 500 fmol. Add 5X TEDA to it’s 1X working concentration and incubate at 30C for 40 minutes. Transform 2 uL in 100 uL of competent cells and screen colonies!
I have been slowly trying this method side by side with Gibson assembly and after having success about a dozen times it has now completely replaced it. Essentially you are relying on the T5 Exonuclease to chew back the DNA ends, they anneal to their partner, and the E. coli actually repair it once transformed. I’ve TEDA’d easy 2 fragment assemblies, including inserting Mashup-RT into it’s vector, as well as gnarly 4 fragment assemblies of viral genomes.
Anyway, if you love Gibson assembly you will love this protocol, give it a try, you likely have everything you need in your freezer right now!!!
This post is awesome. Just need one precision. You stock the 5X TEDA Cloning Mix at -20oC or -80oC?
Hi, the original paper says you can store it at -80 or -20.
https://academic.oup.com/nar/article/47/3/e15/5193337
Super cool!
I`ve tried SLiCE. I worked but i did not got a high efficiency.
I`ll definitely try it, but I’m stuck with Gibson when cloning direct in Bacillus. It is impossible to transform using a nicked DNA.
It’s been some time since I have posted here so hi everyone 🙂
I think this also needs mentioning…
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0137652
It just works, lower efficiency but just works, especially good for one or two fragments (in my experience) – just purify fragments, mix with water and transform.
Cheers!
I can’t repeat TEDA protocol, although I’ve tried three times on February 2019.
But, I’ve succeeded many times with Hot Fusion protocol. (Works very WELL!)
Hot Fusion Cloning: (Like Gibson BUT NO Taq ligase=>It’s the most expensive)
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115318
AQUA Cloning also works, but lower efficiency. (in vivo assembly by E.coli)
https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0137652
HAC Cloning also works, but lower efficiency too. (Very Very QUICK!!)
A little like SLIC……
https://peerj.com/articles/5146/
Thanks a lot for the feedback, I’m sorry it didn’t work for you, I’d help troubleshoot but whatever gets the job done is fair game. With this type of overlap-based cloning I think different techniques have different preferences for stuff like length of overlap, sequence of overlap and surrounding area…also I would say for more difficult assemblies, maybe more than 3, some mixes are better than others.
And thank you for the list of cloning methods! I will be tryng the ligase-less one.
Greetings,
In your opinion, would Hot Fusion work with homemade phusion? Then it will be a cost-cutting initiative 🙂
ML
Absolutely! It should work a treat. I would see these mixes as a continuum with TEDA on one extreme of simplicity and Hifi mix as the extreme of complexity. Adding homebrew phusion would spice things up, for sure.
Reading the comments above, I wonder if it didn’t work for some, is it because the T5 Exo was at very low amount? looking at the protocol 1 µL of T5 (10U) in 1ml 5x rxn buffer, and use it at 1X.
What’s your experience with this Alex?
Ryan
Hey Ryan,
The beauty of the TEDA method is probably the simplicity and cheapness of the method. Yeah, the 1 uL in 1mL seems crazy low but it repeatedly works for us. The only other thing I do is make sure the DTT is freshly prepared that day. Also we’re using Fluka brand PEG8000 from the dark ages, does brand make a difference? Eh, I’ve heard mythical stories, but maybe it’s old timers brand loyalty left over from when they were PhDs…it will be a dark day when we reach the bottom of that jug, I don’t think Fluka is a company anymore lolololol
Our experience is that TEDA is awesome for 2 fragment assemblies (i.e. 2kb insert + 8kb vector) and pretty good for 3 fragment assemblies. With 4 fragment assemblies your efficiency plummets…you can still pull it off, but at that point the cheapness of the mix is probably offset by the low efficiency, that’s when I’d pull out the homemade gibson or NEB’s Hifi mix.
I wonder if adding ET SSB (Extreme Thermostable Single-Stranded DNA Binding Protein) would improve the efficiency of the TEDA method as it seems to for Gibson Assembly?
https://www.biorxiv.org/content/10.1101/2020.06.14.150979v1.full
Hehehe, I did play with this just a little bit when I first read about TEDA. I added a small amount of SSBs to the mix and compared them. I didn’t see a huge difference with and without SSBs. I would say I likely didn’t use enough to have much of an effect.
I didn’t go further down that road because the main advantage of TEDA is the cost. Once you start adding other enzymes you’re slowly recreating Gibson mix, you know?
But yeah,TEDA is a good base you can build on, add whatever you have in your pantry.
Hi Alex,
Thanks for sharing this.
Do you know if this protocol will work with transformation by electroporation or if chemically competent cells will be needed? In my experience, in-fusion cloning did not work with electroporation.
Ingo
Yes, you can use TEDA/Gibson for electroporation, however you need to be careful to only use 1-2uL of reaction per ~40uL of cells (at least for agrobacterium). The additional salts and components of these mixes make it more likely you will get arching. I used to electroporate 3-4 fragment assemblies, so it is possible. You can also purify your TEDA/Gibson reaction with a silica column before electroporation to be able to jam more DNA into your cells.
I got your email btw, sent you a reply 🙂
Hi!
I got a lot of false positive with TEDA. I did it with only the vector and compared it to the vector and insert and I got the same number if not more colony with the vector alone. I was wondering if I’m the only one with this problem.
I’ll be publishing a video on screening these types of overlap cloning techniques in a few days, but it’s a fairly common problem. How many fragments are you doing? High background on the control plate could indicate insufficient DpnI treatment and/or too much template DNA used during the initial PCR. Start with troubleshooting those issues, unfortunately if that’s not your issue then it gets a bit more complex, there are constructs which can be toxic to E.coli, so fewer colonies on your experimental plate could be a reflection of what you’re trying to make. You can get in touch through my contact form and I can help more specifically if you need.
Only one fragment and the vector.
Assuming they’re pretty standard size fragments like a 5kb vector and a 2kb insert, then a 1:3 vector:insert molar ratio should do the trick. If the insert is under 500bp I’d do a 1:5 or 1:10 ratio.
How much input plasmid did you use in the PCR to generate your vector and insert? Keep it under 1ng for a 25 uL reaction so the DpnI can do its job easily, otherwise your cloning may have worked but you just have tons of background.
Gel purification of products is the best way to go before the assembly reaction, and a silica column purification is okay if you have a reasonably clean PCR product.
How many fmol of vector and insert did you use? 50 fmol of all fragments is doable but grim, 200 fmol is ideal.
Hope some of that helps with troubleshooting, if you give me more information I can help more.
Hi!
I had tried this protocol many times and it works excellently well!
I wonder how it performs when more than 5 fragments were in one pot, and will there be some mistakes in the connector between fragments?
I personally havent seen errors in overlaps yet, but I try to avoid designing them in overlapping regions where they might slip.
I have used TEDA up to 3 fragments successfully, over that and I get lower efficiencies. Usually I switch to Gibson then.
Hey everyone,
we tried TEDA Cloning and Hot Fusion and both methods work like a charm. They even outperform our (quite old) Gibson Mastermix.
Thanks for this post!
Greetz
Wade
Hi everyone,
Thanks for the post. TEDA could work with a digest linearized vector?
Best,
Tasos
Should work nicely with that 🙂
Hi,
We’ve been getting TEDA and hot fusion running in our lab. It seems we’ve had some problems making the 5x cloning mix, as when we use 5x mix made in a different lab in our department, it works well, but when we make our own 5x mix, it doesn’t seem to work. All our MgCl2, TRIS, DTT etc was made fresh…
Any ideas as to why this might be happening? Is the PEG8000 sensitive to degradation?
The two things I would check are the t5 exo and the brand of peg used, see if you can borrow a bit from the other lab.