TOPO TA Cloning – Adding 3’A Overhangs

The TOPO TA cloning kits for subcloning offer an easy way to subclone effectively, provided you can get it to work for you.  The topoisomerase I in which the kit relies on requires the presence of 3’A overhangs on the DNA inserts in order to catalyze the reaction joining insert to vector. Ironically, the enzymes and other components required to add the 3’A overhangs are not supplied with the kits and the protocol provided in the instruction manual is not what I consider ideal.

I have personally never followed the 3’A overhang procedure set out in the product manuals provided; instead, I used my own, which I believe works out more efficiently.

The following is a quick and general run-down of how I clone using the TOPO TA subcloning kits. The focus will be on the addition of 3’A overhangs.

Insert Preparation

* Setup PCR reactions to amplify your insert. Use a proofreading DNA polymerase.

* Run your DNA gels and cut out your insert. If you do not want any possibility of point mutations or DNA breakage, try excising your bands without exposure to any UV.

* Gel purify your gel cut-outs. I recommend using a kit such as Qiagen's QIAquick Gel Extraction Kit. Elute/resuspend the DNA in nuclease-free water.

Adding 3’A Overhangs

Proofreading DNA polymerases have 5' to 3' polymerization and exonuclease activity as well as 3’ to 5’ exonuclease activity (proofreading). It is the 3’ to 5’ exonuclease activity of a proofreading DNA polymerase which enables it to remove any base-pair mismatch, including any overhanging bases, thereby generating blunt-end PCR products. In contrast, Taq DNA polymerases lack the 3' to 5' exonuclease activity, so while Taq enzymes are not suitable for generating inserts for cloning, they are useful for TA cloning for the addition of 3’A overhangs.

* You will need a Taq DNA polymerase which does not have 3’ to 5’ exonuclease activity. Check the product information sheet. An example of such a Taq is Thermo Scientific's Red Hot Taq DNA Polymerase.

* Using the Red Hot Taq as an example, set up the following:

For x1 reaction:

10x PCR Buffer à 2.5ul

MgCl₂ à 2ul

dATP (10mM stock) à 0.5ul

Red Hot Taq à 0.1ul

Insert DNA (from gel extraction) à 19.9ul

Incubate in a PCR thermal cycler à 72 degrees for 30 minutes. Do not cycle. Cool on ice or 4 degrees when complete.

DNA Precipitation

Cool your reaction on ice and proceed to precipitate your DNA inserts.

* Take the above 25ul reaction and add 2.5ul (which is 1/10^th volume) of 3M pH5.2 NaAc (sodium  acetate). Tap or gently vortex to mix.

* Add 62.5ul (which is 2.5 volumes of ice cold absolute ethanol). Tap or gently vortex to mix.

* Incubate the entire reaction on ice for 30 minutes.

* Centrifuge at 16200xg for 20 minutes.

* Aspirate the supernatant and wash the pellet with 500ul of 70% ethanol.

* Centrifuge at 16200xg for 5 minutes.

* Aspirate the supernatant, air dry the pellet and resuspend in 10ul nuclease-free water.

* Use 4ul for TOPO cloning reaction.