I have done TA cloning using PTZ57R/T vector, positive Control with PCR insert(kit) and TA vector is getting transformed but I think, my amplicon with PolyA tailing is not getting ligated. I am not getting any colonies after transformation.
I have carried ligation reaction at different temperatures with 3:1 AMPLICON: VECTOR ratio like 22 for 1hr,4c and 16c for overnight, did transformation and didn't get colony.
I have carried polyA tailing by adding 10mM dATPS to the PCR product with out purification of the amplicon on gel.temp 95C for 10min and 72c for 20min. I have used taq polymerase for PCR. So where am I going wrong?
I see that the problem is with your A overhang protocol. Incubation at 72°C for ~30 minutes is enough. No need to do denaturation. Purify the PCR product and ligate.Before transformation deactivate your ligase enzyme.
First you have to confirm that your ligase is working properly or not and which type of polymerase you are using. There are several types of polymerase which creates cohesive ends and some blunt ends only. You have to do A tailing with your gel eluted amplicon. In uneluted PCR product there can be a chance of ligation of non specific amplication product which can compete with ligation of your specific amplicon. According to size of amplicon you can increase the time of ligation. may i know the size of your amplicon for ligation.
I do a lot of TA cloning w/o problem. so, few points:
For A tailing, I do 2minutes in ice followed by 10 minutes at 72°C
PCR purification
Ligation 1 to 3 ratio (Be careful, the ratio is for the NUMBER of molecule and not the quantity, example: vector: 3000 bp,insert: 1000 bp so you mix 50 ng of vector and 50 ng of insert but if the vector is 3kb and the insert 3kb then you add 50 and 150 ng respectively.
If you're using column for purification, be careful to remove all the wash buffer after last wash, for this change the collection tube, turn 180° the column in the centri and do a 2 minutes empty spin.
quantiufication: don't use nanodrop (not exact), if u have qbit, it's the best. If not quantify on gel after a brief migration (about 1 cm) and with a DNA you're sure about the concentration (load 50 ng of this)
Ligation: Ligation buffer has to be aliquoted. It should smell DTT if not, it's not good anymore. Thaw it 10' before, vortex it strongly before adding to the ligation mixture.
Before adding ligase, vortex strongly dna/ligation buffer mix. then add ligase
good luck
From your note, I see that PCR products were directly ligated to the dT vector. This may be your problem. This is because PCR components or by-products of amplification reation will inhibit ligation reaction. Therefore, the PCR product with or without a dA tailing step has to be purified prior to a ligation reaction. Ideally, PCR fragments should be gel purified. This will also eliminate ligation of non-specific low molecular weight fragments as stated in previous answers. Purification by precipitation of PCR product by addtion of 2.5 volume of ethanol also works well if amounts of specific product are sufficient.
Second, low amont of PCR product could be a problem ,In this case, ligation effciency can be increased by extending ligation time - reaction over-weekend at 4C works well.
Our lab has had luck performing A-tailing of the PCR product without purification, however the polymerase you are using can have exonuclease activity as mentioned previously which might remove any "extra" added bases, i.e. your A-tail. We have directly used PCR product amplified using Thermo Fisher's DreamTaq green PCR buffer/polymerase (https://www.thermofisher.com/order/catalog/product/EP0711). When in doubt, you can reserve an aliquot of your PCR product and perform A-tailing and ligation of both purified and unpurified PCR product in parallel and compare the results. When purifying, you can use various methods of purification such as gel purification and extraction (using an old-school lab-made protocol like what can be found in "Ream, W.; Field, K. G. Molecular Biology Techniques: An Intensive Laboratory Course, 1st ed.; Academic Press: San Diego, 1999."), using a Qiagen gel extraction kit after running your PCR product on an agarose gel (https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/dna-clean-up/qiaquick-gel-extraction-kit/#orderinginformation), or using a Qiagen Minelute PCR product cleanup kit (https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/dna-clean-up/minelute-pcr-purification-kit/#orderinginformation) to clean up your PCR product, however in general unless there are specific problems you encounter, you could theoretically get away with directly using your PCR product. (You would need, however, to run a gel and estimate the concentration of your PCR product using a molecular marker with known quantities of DNA, such as the NEB 1kB Ladder, or a DNA sample with a known concentration to compare your product and calculate a 1:3 vector:insert ratio.) Furthermore, our lab has not had any success with the "rapid ligation protocol" using T4 ligase that NEB supplies with the enzyme; overnight incubation @ 4 degrees C in our cold box is all that works for us. In terms of further clean-up, you should heat-deactivate your ligase @ 65 degrees C for ~10-15 minutes before transforming E. coli with your newly-ligated plasmid and insert, however there is no real "clean-up", per se, after A-tailing.