TA Cloning - (Mar/14/2015 )
Currently using TOPO cloning kit from invitrogen and there's no colonies growth observed on ampicillin plate.
The vector and PCR products should be fine.
Was wondering if it could be due to the ratio of TA vector: insert.
I amplified my PCR products by using Econotaq polymerase kit, then purify it using promega wizard purification kit.
The yield of my purified pcr products is about 100 ng.
I put about 200 ng of PCR products into the TA cloning reaction with only 10 ng of TA vector.
My question is:
Supposedly more PCR products (inserts) the better for TA cloning or should I stick with the standard 1:3 ratio?
Can you use traditional restriction enzyme digest + ligation?
I've only tried those TOPO cloning kits a couple of times and not been too impressed. If it didn't work the first time, no amount of trouble shooting fixed the problem. I just figured out how to do with RE+digest, or ended up buying the damn gene.
Consider how long it'll take to figure out the problem, the cost of your time, and the cost of all the reagents, then look up the cost of buying the gene. You could be surprised.
I've tried the conventional method a few times but ended up with failure, it seems like the RE cutting site of the gene is too towards the edge and would improve tremendously if it's first ligated to a TA plasmid first before digested with RE and ligate with another plasmid of my interest.
Meg P. Anula on Sun Mar 15 07:04:37 2015 said:
I've tried the conventional method a few times but ended up with failure, it seems like the RE cutting site of the gene is too towards the edge and would improve tremendously if it's first ligated to a TA plasmid first before digested with RE and ligate with another plasmid of my interest.
I'm unsure what you mean by "RE cutting site of the gene is too towards the edge" .
Do you mean there is a cut site at the end of your gene that you also need to use to ligate into the vector?
If so, then there is a way around that with overlap extension.
Amplify from 5' end of your gene (maybe slightly upstream of it) all the way to cut site near the end of your gene using a reverse primer that removes said cut site but maintains amino acid sequence.
Also amplify in a separate reaction from your cut site at the end of your gene using a forward primer that removes said cut site (reverse complement of the one used above) to well beyond the end of your gene, that is several hundred basepairs past your gene of interest.
Gel purify both PCR products, and fuse using overlap extension. For this PCR, use forward and reverse primers that exactly flank your gene of interest (nested primers) and also contain the appropriate restriction sites for ligation into your plasmid.
For this overlap extension PCR, use ramp the temps slowly as described here.
I've never seen TOPO kit fail. You don't even measure the PCR product concentration or worry about some ration, and just use some ul form reaction as specified in the manual.
But it is crucial to have the right combination of kit and polymerase (Taqs for TA and profreeding polymerases for Blunt), but that seem to be OK.
PCR product should be fresh, you don't need to purify it, but prolonged strorage or further manipulation may degrade the A overhangs. Without them, there is no ligation.
Did you tried the control transformation to be sure bacteria is all right?
And if I'm not mistaken the kit also contains control template and primers to check out the whole PCR process before ligation.
Anyway is there a reason why you don't do it as in the manual (I have TOPO TA cloning kit for sequencing, but the ligation should be the same), just use fresh single band of PCR product to ligation reaction?
The kit states that purification of PCR products decreases efficiency.
I had a difficult gene I was cloning not too long ago...6.5kb with 70% GC content and more than a dozen repeat sequences in it...PCR worked great. Tried to gel purifiy insert, tried to do just a PCR cleanup, tried adding PCR mix directly into ligation mix, tried the commercially prepared competent cells that came with the kit, tried my own cells, tried electroporation...never got the TOPO kit to work. Spent $300 on new kit...same results. Got plenty of colonies but none with the insert. Too many false positives. Tried it 20+ times. Checked hundreds of colonies by both colony PCR and miniprep/analytical digest. Positive controls worked. Probably spent $500 on reagents to do this with nothing to show for it.
Then I went back and figured out a way to get it in via traditional restriction/ligation. Took a couple tries but at least I was able to troubleshoot it that way. Used probably $75 worth of reagents.
I generally dislike kits because it fosters a sense of carelessness in students' not understanding what they are doing. They just mix and hope for the best. And if it doesn't work, then the project stops before it starts because the troubleshooting ability of a kit is too one dimensional. It's like pulling teeth to get a student to even read the manual for most kits.
That's just my biased opinion tho. 
With difficult templates, things are just you know.. difficult ;)
Actually kits are you know, for that kind of use, when you just want to simply clone your fragment, because your real aim is not cloning or learning how to became a superskilled bacteria guy, but when you want a result as fast an comfortable as possible.
That's why I use the best PCR master mix available, because I make new PCR reactions like every other week and I just don't want to spend my time optimizing something I may run 10 times at all. It's different if you want to make a super cheap optimzed reaction for screening or routine something. It's just that optimizing PCRs is not your research aim, PCR is just a way how to get there. Same is for cloning, if it's not some hardcore construct preparation, when you need half a year of no results to get the mad skills.
Of course relying solely on kits is stupid, you need to read the kit, understand how it works and at what cases it won't work and see if it's even suitable fo you. Kits are comfortable but they are not no-brainers. You still need to think and understand it to work with it.
I don't think you only have option to stupidly mix kits and if they don't work then stare into the walls or do everything the "right" harder, classic way. You should be able to do both and able to decide when to use what (by the way I woudn't buy the same kit if it failed once.. ;) at least Invitrogen has Zero brand of vectors, that minimize false positives, if nothing, but given your high GC product.. I would suspect it to make problems and try to find something about it before trying new kit, I like kits but I care about the money as well ;))
And by the way, as I was googling for something about high GC and TOPO, I found this..
So it seems that Econotaq doesn't even add A overhang, but a G. That will never work with TOPO TA cloning (unless, maybe.. if you added some normal Taq to the mix..)
http://lucigen.com/GC-Cloning-and-Amplification-pSMART-GC-Vectors/
It seems Lucigen has it's own cloning patents based on this overhang, but hey, the NEVER mention it in the manual, that it doesn't make A overhangs but G, and they call it a "Taq"!
I would tell them to go to hell for that, really.. great for the unaware customers. Screw them..
Thanks Trof I appreciate your thoughts. I agree with what you say.
No doubt kits have a time and place when you just need to get to the next step and be able to do the actual experiment you intended to do in the first place. In my last post I was probably a little harsh on that simply because I've been trying to teach some new rotation students in the lab and they are struggling to do some basic cloning. And when I ask them what they are trying to do, it becomes evident that nobody in their first two rotations taught them anything about what they are doing...rather just to add X ul of A to X ul of B, wait X min and poof it should work. And when it doesn't work, they blame the reagents or the protocol or anything other than their own hands. I get frustrated by that. But maybe I need to be better at learning a little patience as well. :)
Trof on Fri Mar 20 19:08:40 2015 said:
And by the way, as I was googling for something about high GC and TOPO, I found this..
So it seems that Econotaq doesn't even add A overhang, but a G. That will never work with TOPO TA cloning (unless, maybe.. if you added some normal Taq to the mix..)
http://lucigen.com/GC-Cloning-and-Amplification-pSMART-GC-Vectors/
It seems Lucigen has it's own cloning patents based on this overhang, but hey, the NEVER mention it in the manual, that it doesn't make A overhangs but G, and they call it a "Taq"!
I would tell them to go to hell for that, really.. great for the unaware customers. Screw them..
Thanks so much for the info. I'd never know it because in their manual they never mentioned about this specialty of their taq polymerase. It's truly upsetting because I've wasted quite some times troubleshooting. I guess it's part of my fault for not knowing much.
Anyway, I actually performed a control accordingly to their manual and turn out this TOPO vector (purchased over a year) is not functioning either. I've double checked my competent cells to make sure they work and they did. So for me it's very likely something is wrong with the vector.
The reason I stick with the EconoTa master mix is because they worked well for one of my (difficult) gene. For which I had done it manually and tried to optimized the MgCl, but still for that particular gene, it have got unspecific bands. Not the case with EconoTaq, the mix is just so optimized that I'd get only one specific band.
I think I'll have to purchase this GC vector.
Anyway, I have a question in mind, since this Econotaq polymerase master mix I used have this green dye in it, would it be okay not to purify it before i use on GC vector? Since you said purification can degrade the A-T overhangs so i wonder if it'd do the same for G-C as well.
Thanks.