A-tailing should be sufficient to allow incorporation of your qPCR product in to an Easy Vector system.  I'm assuming your ligation reaction is happening at RT for 30 mins to 1 hour.  Maybe perform the ligation over night at 16oC.  Or try a new ligase.

Be sure you're using dATP and not dNTPs in your A-tailing reaction.

Also use a decent quality Taq DNA polymerase.  I used to use Fermentas Taq and always had horrible results.  The second I switched to Takara ExTaq I never had problems.  I suspect other suppliers like NEB will have good quality enzymes as well.  

If you're having problems, be sure to use blue-white colony selection as well.  

Many thanks. Dylan. I did the ligation at 4 degrees overnight, but only got 2-3 blue clones. The competent cells are good. I heard from internet the company will add something in SybrGreen mixture to avoid problems during qPCR. The added stuff is incorporated into DNA and is toxic to E.Coli. Do you have any idea about this?

Hi, 

If it is still fail ligation into vector, maybe need to do PCR clean up to solve DNA in H2O. All of these step should ASAP, before A-tailing is release. 

If it still not work, I will use 1~2ul qPCR product to do another PCR using normal Taq. It would only need 5~10 cycle and should see strong band in agarose gel.

Hope it would help you. Good luck

One reason could be the product size.In general the product size for a SYBR green assay ranges from 80-150 bp. However exceptions are there.I have seen some TA  cloning kit starts from 400bp.

Hey,

I will tell you how to do it.

1-perform say 25 ul PCR reaction with Taq polymerase (will add overhangs fo TA cloning)

2-run say 5 ul on the gel to ensure that the desired band is present

3-use remainings of your PCR reaction directly in clonig reaction without any purification

if there is a single band, it works

easy, cheap, working procedure

Hi Peijin,

I would first verify that your ligase operates at 4oC.  That might be your problem.  In my experience I have never run a ligation at 4oC.  Most commercially available enzymes suggest working between 12-16oC for overnight ligations.  Invitrogen's T4 DNA ligase, for example, suggests 16oC for 24 hours if you're trying to ligate blunt ended fragments.

Oddly enough I ran in to something exactly like what you're hypothesizing last summer while trying to clone some genes.  It was Safe-T-Stain from Bioshop - an ethidium bromide alternative. I showed in my troubleshooting that this product, Safe-T-Stain, completely inhibited ligation reactions as well as E. coli transformation.  The first was shown by trying to amplify across the multiple cloning site, the second shown by transforming uncut vector.  Both experiments were performed with DNA which had been gel purified from gels containing Safe-T-Stain, or SYBR Safe DNA stains. SYBR Safe treated DNA worked fine, Safe-T-Stain treated DNA couldn't be ligated in to vectors, and uncut vectors couldn't be transformed in to E. coli.

If you think your problem is associated with a component of the qPCR master mix, then I would run a standard, non-qPCR reaction, harvest the amplicon by gel purification and try to clone that.  Be sure to use a non-Taq enzyme for the purification, then A-Tail etc.  That way you're as close to your qPCR conditions as possible.

Best of luck. 

Don't forget, qPCR uses dUTP instead of dTTP (or sometimes a mix of the two). Type B polymerases (phusion, iproof, vent, etc) don't play nice with dUTP.