We worked with the TA cloning and TOPO cloning kits of Invitrogen, as
well as the PCR-Script of Stratagene. The first two take advantage of
the 3'-end A-overhang, added by regular Taq polymerase to many of the
amplified products, in addition the TOPO is faster, only 5-minute
ligation. However, we did not try them with Vent, and I think there
might be a problem with Vent, because if I am not mistaken Vent does not
produce A-overhangs (or produces them much less than Taq).
The PCR-Script kit of Stratagene does not need overhangs, it should work
with the Vent and any other thermostable polymerase. The disadvantage
of the kit is that it produces (in our hands) lots of background - we
had up to 30:1 ratio of clones with inserts vs. religated empty
vectors. The advantage is that the ligation is done for one hour at
room temp and the SrfI restriction endonuclease is present in the
ligation mixture, cutting all empty vectors that religate, therefore
there is no need for dephosphorylation of the vector or phosphorylation
of the primers.
I must say that we prefer cloning with no kits, we incorporate cutting
sites at the 5'-end of our primers and ligate (sticky ends cloning) at
room temp for one hour. Blunt end cloning is also at room temp, for 2
hours, the primers are phosphorylated prior to the PCR reaction
(single-stranded DNA works much better with the PNK than a PCR
product). All our vectors are dephosphorylated (even for sticky end
cloning, I can elaborate why if needed).
It is not the cloning kit, but the competent cells what makes the
difference and matters to us. We prefer ultracompetent cells from
Stratagene, and we found out that they could be frozen at -80 and thawed
on ice a couple of times without noticeable loss of competence.
There is a 5-minute ligation kit from Boehringer, if you just want to
cut the time for ligation down. However, you still have to spread your
cells and wait for the colonies overnight, therefore I do not see the
big advantage of 5 min versus 1-2 hour ligation.
Best regards,
victor
www.alphadna.com
____________________
Susanna Sroka wrote:
Can anyone recommend a PCR cloning kit that will work for Vent amplified
PCR products? I'm unfamiliar with these kits, but have recently heard of
them. They sound great and will reduce ligation and transformation time.
Susanna Sroka
Samuel Lunenfeld Research Institute
Toronto, ON