Hello, I would suggest that you create the restriction site by designing in
the overhangs. The likelihood of getting poor yield goes up dramatically
with each isolation step. The key to the overhangs would be to phosphorylate
the primers to allow ligation. Designing in an identifying characteristic
would be an excellent idea as well. IE: If you are able to destroy a unique
restriction site in the vector (+target sequence, if it is already in) by
ligating in the adapter, or creating a new unique site (same constraints) you
would be better off. Alternatively, if the new sequence is unique enough to
allow PCR, use a second set of shorter primers that don't have the
palindromic 3' ends (if possible) to screen for the insert/allow non-target
insert known PCR site. If you can't afford so many primers, use the full
length adapter primers. Good luck, Warren BTW: Watch out for frame shifts,
inadvertant termination signals, and typos. (Guess how I know...)
>In article <86nnan$tmo$1 at nnrp1.deja.com>,
>jen_the_glick at my-deja.com wrote:
> Hi. Have two 40 bp, complimentary oligos that I am annealing to one
> another. They have restriction sites on the ends, so after annealing I
> am cutting with the enzymes, heat-inactivating the enzymes at 65 C for >20 minutes and then ligating. I am heat-inactivating the enzymes >because resolving them on a gel and gel purifying them seems awfully >difficult for DNA this small.
YES IT IS! Barring acrylamide/ 5%+ agarose, you will have a difficult time
of it. That's kind of going back to the days of expensive/difficult to
For complimentary oligos at high concentration, the annealing should (for the
most part) happen at RT, semi-instantaneously. If you are working with a
single stranded target, there is little if any competing dna to block the
annealing site. Working with M13 based ss dna sequencing is much easier than
ds sequencing (that's why M13 sequencing was developed earlier than ds. Or,
I should say, that was the only protocol I used from 84-87; Remember Klenow
> The annealing protocol I am using is mixing them in equimolar amounts in
> the presence of a little bit of salt, heating to 90 C in a PCR machine,
> then cooling at a rate of 1 degree/min.
>> So when I heat-inactivate my restriction enzymes, should I be cooling
> very slowly, as I did before? Otherwise, might I be undoing the good I
> did in the initial annealing reaction?
>> Sent via Deja.com http://www.deja.com/> Before you buy.
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Before you buy.