Re: [DIYbio] Chloroplast export peptide

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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