topo TA cloning kit (invitrogen) - URGENT (Jul/09/2002 )

I have been trying unidirectional topo TA cloning kit from Invitrogen several weeks, and I have not get any positive result. I have beeing using a mixture of enzimes (Taq platinum, pfu, X-ACT...) with subsequence purification and incubation with Taq for the A-overhangs. Nothing resulted.
Which are the best enzimes for this cloning kits?
PLEASE, ANSWER NOW. THANK-YOU

Got me - I clone with the Topo TA kit all the time with excellent results. The only Taq I use is Gibco (I guess Invitrogen know) Taq DNA polymerase (18038-042). Usual recombination percentages are at least 70% and often 100% take the vector.

I use the TA and TOPO cloning kits from Invitrogen. I use Promega Taq for the TA and mix about 1ul of neat PCR product (25ul, 30 cycles) in the ligation mix. For the TOPO kit I use Promega Pfu with a little extra Mg to help the PCR. If you need A overhangs for the TA (i.e. you used Pfu for the PCR), I add some fresh Taq (0.1ul per 10ul PCR reaction) and come 2mM ATP to the PCR reaction after it's finished (so it can still use the buffer in the reaction) and cycle at 94oC 20 mins, 72oC 20 mins. THEN run the PCRs to check, leaving a little left over for the ligation. You can always put in as much PCR product as possible into the ligation (about 6 ul) to increase chances of it working. THE TRICK IS TO ADD THE A'S AFTER PCR BUT BEFORE PURIFICATION OR THERE IS NO BUFFER TO MAKE THE TAQ WORK. Hope this helps, email me if you have any questions.

Ainara,

Used Takara ex Taq polimerase, is high fidelity, it will the AAA overhands. Or ohter polimerase that add the 3' AAAA.

Do your PCR and add 10 minutes at 72 C for 10'. ( Last extension)


Purified your fragment and Cloned with a TA Cloning Kit (plasmid with TTT over hands)


Good Luck,

agustin



I have been trying unidirectional topo TA cloning kit from Invitrogen several weeks, and I have not get any positive result. I have beeing using a mixture of enzimes (Taq platinum, pfu, X-ACT...) with subsequence purification and incubation with Taq for the A-overhangs. Nothing resulted.
Which are the best enzimes for this cloning kits?
PLEASE, ANSWER NOW. THANK-YOU

-Ainara-