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iTAQ and TOPO TA Cloing - (Oct/06/2006 )

Hi, does anyone know if I can apply iTAQ (from Bio-Rad) PCR-ed products for subsequent TA cloning using TOPO technology from Invitrogen?

Many thanks!

-Biomedmedia-

QUOTE (Biomedmedia @ Oct 6 2006, 07:27 PM)
Hi, does anyone know if I can apply iTAQ (from Bio-Rad) PCR-ed products for subsequent TA cloning using TOPO technology from Invitrogen?

Many thanks!


Why not answers??? Please do help!

Thank you!

-Biomedmedia-

possibly because nobody has experience using that combination before. The TOPO kit comes (if I am not mistaken) with it's own taq enzyme.

Anyhow, I was unable to find out if iTAQ has been modified to include proofreading capability. It does have an antibody binding for hot start capability... but if that is it's only modification then it should be possible use iTAQ for TA cloning.

I would say, give it a try and see if you get any white clonies with the insert.

-perneseblue-

QUOTE (perneseblue @ Oct 11 2006, 01:39 AM)
possibly because nobody has experience using that combination before. The TOPO kit comes (if I am not mistaken) with it's own taq enzyme.

Anyhow, I was unable to find out if iTAQ has been modified to include proofreading capability. It does have an antibody binding for hot start capability... but if that is it's only modification then it should be possible use iTAQ for TA cloning.

I would say, give it a try and see if you get any white clonies with the insert.


Thank you again, perneseblue!

I did try to use iTAQ and there is no single colony, although biorad support team told me it should work for TA cloning. iTAQ does not have proof-reading capability. I am going to use Invitrogen's Taq polymerase this time.

Since my primers start with CACC (initially for directional cloning for expression), I am wondering if this will decrease the TA cloning efficiency. Do you have any idea about that?

-Biomedmedia-

QUOTE (Biomedmedia @ Oct 14 2006, 11:16 PM)
Thank you again, perneseblue!

I did try to use iTAQ and there is no single colony, although biorad support team told me it should work for TA cloning. iTAQ does not have proof-reading capability. I am going to use Invitrogen's Taq polymerase this time.

Since my primers start with CACC (initially for directional cloning for expression), I am wondering if this will decrease the TA cloning efficiency. Do you have any idea about that?



QUOTE (ML1975 @ Oct 4 2006, 03:51 AM)
There is another suggestions. The primers are crucial to whether you get A-overhangs onto your PCR product. Check this paper out... Biotechniques, 1996. Vol. 20(6). pg.1008-1010. You can add a 6-bp sequence to the end of your primers which will give you 100 % adenylation and may help your ligation. The paper also discusses some primer sequences that are resistant to adenylation. Hope this helps.


I don't. However ML1975 has quoted the above paper in a situation similar to yours. Apparently the ends do make a difference. I would suggest you look up that paper.

-perneseblue-

Thanks. I know this paper but since my primer does not start with A, i thought although it's not optimal, it might work. I am testing now. Will let you know the result soon.

-Biomedmedia-

Biomedmedia.
There is differnt kits, TOPO, you can use. Anyway, the A-overhang you are refering to should be added by the Taq-polymerase so you dont have to think about that.

-hollegaard-

Taq polymearase is quite cheap so just add 0.5µl regular taq and incubate 10' at 72° before doing your topo reaction.

and maybe a simple request to roche regarding that point will give correct answer about the capability of iTaq to add single AAAA overhangs.

-fred_33-