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TOPO TA Vector Cloning problem - no colonies - (Aug/10/2005 )

I am Ahmed
My problem is in TOPO TA CLONING Reaction.
I am using TOPO TA Cloning Kit. (Invitrogen).
After one shot chemical transformation and bacteris culture , i kept my Amp. plates overnight. and i didn't get colony in my plates.
so, please anyone know what is the reason??

-Ahmed shafa-

Hi Ahmed,

Did you do the control reactions as suggested in the TOPO kit? Did they work?

At this stage it could be many things - we need more information if we are to help you. biggrin.gif



Thanks a lot for your reply.
No, I did not do the control reaction.

-Ahmed shafa-

Does your PCR enzyme add the extra A overhang? For this you can perform the control reaction as described.

Are your cells competent (check this with the control pUC19 or any other plasmid).

Is your medium ok? Check this by just putting cells on there without transforming them with DNA.

You are 100% sure about adding amp and no other antibiotic? (Can be checked by transforming one plasmid that does have AmpR and another one that doesn't).


The control reaction not provided in my Kit.
I suspected the media. i think there is something wrong in LB agar plates. so i prepared another one .. but colony growing.

-Ahmed shafa-

I have been using the TOPO TA kit to clone in the past few months.

First of all I would suggest downloading the instruction manual from their web-site, it doesn't come with some kits. It will tell you how to set-up your control reaction - with the control DNA and control primers that have to be part of ur kit!

Also, perform a control from the transformation using the pUC19 DNA they provide. It helps in letting u know if ur cells are really competent.
Mine weren't and in talking to a technical representative I also learnt that u should still use a 3:1 ratio from ur inster:vector ligation. Run out ur PCR product on a gel with a ladder that can be used to quantitate ur DNA amounts. My most recent transformation I had to dilute my PCR product 10 fold and used 2ul of this in my ligation.
I finally got colonies afer 2 attempts - 1st time too much PCR produt and 2nd time the cells were not competent!

All the best with ur cloning!