It will keep adding; you can try to work out the proper incubation time so as to not add too many (it's not an overly processive transferase). Blocked nucleotides like ddNTPs will likely interfere with ligation; you would have to use a reversible terminator of some sort.
In any event, the ligation of a single dA to a tail of 3 dTs (for example) is probably still more efficient than blunt ends.
I believe the issue disappears altogether if using taq though.
On Feb 2, 2013 9:12 AM, "Cathal Garvey" <cathalgarvey@cathalgarvey.me> wrote:
-- I thought TDT would just keep adding a splurge of random nucleotides? Do
you use blocked nucleotides to stop it going nuts, or is it less
prolific than I thought?
On 02/02/13 09:46, Paul Sebexen wrote:
> The most common enzymes I've seen used for dephosphorylation are
> Antarctic Phosphatase and Alkaline Phosphatase (CIP).
>
> Generally the A- and T-tailing are done with Terminal deoxynucleotidyl
> Transferase. Taq polymerase can also be used (will add A's
> preferentially even if given a mix of dNTPs), but is a bit less
> efficient at T addition as a 3' overhang (requires only dTTP be added to
> this reaction). After the complementary tails are added, you can proceed
> with ligation as normal; the efficiency should be significantly higher
> than with blunt ends and you can relax the insert:vector ratio a bit.
> There are many variations on this - I've even seen protocols using C-G
> tails.
>
> Not requiring directionality is definitely convenient.
>
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