Blunt end cloning is difficult. If you have selective media then you can find some positive clones.

I do have the selective media and since the vector contains both kanamycin and zeocin, I have prepared separate plates containing kanamycin/zeocin individually and together. You are right, I did two successful cloning of patient gene amplified by Taq into TOPO vectors and it worked very great for them but I am fiddling with this blunt cloning at the moment. This gene couldn't be amplified in great amount and required size by Taq. So, I switched to Deep vent.

You could try pJET1.2 vector - we use it for blunt cloning and it works very well (no A tailing needed). For the A tailing reaction maybe the efficiency of A tailing is higher if you use just dATP instead of dNTPs?

Well first I can see the concentration is low. this will dilute the fragments so the change that you blunt ends will interact each other. Then if you can avoid blunt end cloning just do it. if not try to do at least one stick end. But my experience if you must do a blunt end cloning is as follow. use at least 2ug of you vector (in duplicate) and open in the desired site. then Blunted it (take into account some enzymes do 3'end over hand and other 5' ends over hands, this is important because Klenow just add bases in one specific, if not you need to use T7 DNA pol or something like that to blut your ends) or you need to do exonuclase to blut also. For me is better to REMOVE phosphate group with Phosphatase and to have the vector ready to ligate. Then your PCR produc well the proble is simple may be for yoru PCR product you will need then TO PHOSPHORILATE your fragment with a KINASE and ATP, and increase your FRAGMENT concentration by PCR and column purification. after that just ligate in the lower volume, and placing at Molar ration 3 fold more insert than vector.

should work.

To be honest: I don't bother with blunt end cloning anymore. If there are no fitting restriction sites (vector and gene) I create a new MCS into my vector of interest and also add the restriction sites to the PCR primers to add them to my gene. Even if this requires two cloning steps I was always faster with this strategy than with blunt end. The other variant that I really like and provides good success with long and rather difficult to clone fragments is to switch to gibson assembly. There are now also good working commercial kits available.

Hi Shahid,

I've never used blunt TOPO vector, but I've done some other clonings using blunt ends. I've noticed that you are using Vent polymerase from NEB, so maybe your problem is during the PCR cleaning process given that TA-based cloning gives you negative results as well. A simple ammonium-chloride/isopropanol precipitation could clean in a better way your PCR product. Additionally, increase ratio insert:plasmid to 5:1. Good luck!

Im using PFU polymerase for blunt-end PCR products, i m used to add just a little more T4 DNA ligase in reaction mix ( everything else acording to protocol ) time : 16H at 4°C

Why are you cloning "blunt" ? Be more imaginative, try something else!

The easiest way to produce blunt-end PCR products is the use of polymerases that produce blunt ends. Like Juraj Bugala I had good experiences with Pfu-Polymerase.

Try reamplification of your PCR-product with Pfu-polymerade and clean up your PCR-product by agarose gel elecrtophoresis. This additional cleaning step produces better insert DNA than simple cleaning of the PCR reaction with spin columns.

If you have often PCR fragments wich do not "fit" any RE site on your vectors you may consider using the DISEC-TRISEC method http://www.ncbi.nlm.nih.gov/pmc/articles/PMC331481/ with this method you can clone any fragment without using RE digest of your PCR product.

I recall having the same sort of problem in the past. Eventually I gave up on

TA-cloning, built PCR primers with good unique restriction sites on the outside ends, digested the PCR product and cloned into the expression vector digested with the same enzymes. Good idea to add a 3-4 bp to the external ends of the primers next to the restriction sites.

I guess Shahid is following the right protocol when using blunt pTOPO vector to clone his insert (that means no need of ligase) and Deep Vent polymarase (so his PCR product has blunt ends). With all this kits it´s very important how many times you took the vector from freezer and thawed it in order to perform a ligation. I found Zero Blunt pTOPO kit worked very well (many transformants) the first several weeks after receiving it. Then I started getting less colonies. After 3-4 months, I got none or just a few colonies, even for the control TOPO cloning reaction and using new Top10 competent cells. I could get more colonies increasing the incubation time to 15-20 minutes. Again, when I ordered a new kit, I got many colonies with the same insert. Because you´re not going to buy a new kit every time you get low efficiency, try to increase the incubation time and use different amounts of PCR product. You only need a few positive clones. The protocol recommends 4 uL of PCR product at 2 ng/uL, that is 8 ngs, and 1 uL of pTOPO vector at 10 ng/uL. Maybe you´re using a wrong concentration of insert in the TOPO reaction. If you want to change the vector, try pGEM-T and do the PCR reaction with any good polymerase that adds A at 3´-ends. This ligation is less efficient than TOPO one but I found it´s more reproducible for longer periods.

For cloning of blunt PCR products I am not using a vector kit. Instead I cleave a standard vector like pBluescript SK+ with EcoRV and treat it with alcalic phosphatase. If you purify your PCR product e.g. after gel electrophoresis and then ligate it to the prepared vector you can select on ampicillin and do blue-white screening with X-gal for finding the colonies carrying your plasmid. Works best in my hands.

Are you sure about the DNA concentration? The Nanodrop can be quite unreliable. Better check you PCR product on a gel against a marker with known DNA concentration. I'm pretty sure you've also done this, but you should adjust insert and vector concentration according to the manual.

If everything fails, try to purify your PCR product by gel extraction rather than a PCR clean-up kit and elute in water rather than a buffer.

Best of luck!

Allait (use french translator). Je vous donne un tuyau...La "queue" de vos primers vous pouvez en faire ce que vous voulez... Si vous voulez voir comment on peut faire ce que l'on veut en matière de synthèse d' ADN : www.ngyx.eu/products/ngyx-pcr/index.html. Un plasmide de 7Kb avec plus de 100 sites de restrictions et les résistances. Tout exact à la bp près. En 6 étapes de clonage et en 4 semaines... Déposé LMBP-BCCM bien sûr! Je ne vous dis pas comment, à vous de faire marcher vos méninges!

I have regularly generated blunt-ended products using Phusion polymerase which I subsequently cloned into pSC-b vectors (Agilent). It's literally a 5 minute cloning reaction at room temp which you then transform, recover etc. These worked excellently for me. It might work for you too? All the best!

remember your sole objective is to get clones....

Take get good amount of PCR product......primers are generally without 5' phosphate which mean that you PCR product not gonna ligate with each other......make sure the blunt plasmid has 5' phosphate...otherwise no ligation........if your plasmid is unphophorylated then kinase-treat your PCR product before ligation.

every now and then check your ligase activity by inbubating it with sonicated calf thymus DNA or restriction endonuclease generated ladders (donot used PCR generated ladder......one again they would be unphophorylated and cant ligate). after ligation run both ligated and unligated DNA on gel. lagated DNA showed run a big blob of DNA.

Did u do PCR clean up before cloning? also its probably beter to use dATP instead of dNTP for the addition of A overhang

Make sure that the competent cells are OK, try to use some control PCR products to check that the TOPO cloning kit is OK. I dont see the point in doing A-tailing for blunt PCR product cause there are a lot of Blunt PCR product cloning kits that work fine. I use pCR-Blunt vector and Zero blunt PCR cloning kit. It works perfectly, you can reduce lidation volume to 5 ul and use any regular T4 ligase if u run out of the one that is supplied with kit.

Cheers everyone for their valuable input and it helped me a lot to figure out the issue. In the end, I re-amplified my Deep Vent polymerase amplified blunt PCR product with Taq polymerase by performing another round of PCR and I got the desired band amplified in quite good amount that can be utilized directly for TA cloning. Thanks, once again.

Dear Shahid

If you are using polymerase which has proof reading character as in Deep Vent, Phusion etc and trying to clone your PCR product (blunt) in TOPO vector (Blunt-dephosphorylated) so one of your entities(PCR or vector) need to be phosphorylated to get ligated. You can not phosphorylate blunt vector b/c it will be self ligated hence you should phosphorylate your PCR product and try cloning your product you will get +ve clone definitely. I use this commonly in my routine cloning. All the best.

vishal

Just simple. TA cloning vector. 

after the PCR clean the DNA, then add Taq polymerase with a little bit dATP, and put one step at 72°C then Taq will do A tailing. after that it will be very easy to clone into any topo TA vector.

if you can avpid blunt end cloning is best.

you can always add into your primers at 5'end cloning sites too.

good luck

abel.

I wonder if anyone know a blunt end cloning kit that does not contain a T7 promoter. I would like to encode this promoter in my cloned fragment, and only allow transcription from that direction.  

Agree with Vishal Gupta. Thank you for sharing your knowledge