Make a d-strand from ss oligonucleotide ?
andres at calvin.jci.tju.edu
Wed Nov 24 15:28:24 EST 1993
In article <01H5MN7NHCOI9N5SEV at UCBEH.SAN.UC.EDU> WANGNN at UCBEH.SAN.UC.EDU writes:
>From: WANGNN at UCBEH.SAN.UC.EDU
>Subject: Make a d-strand from ss oligonucleotide ?
>Date: 23 Nov 93 01:24:22 GMT
>I am going run the gel retardation to show the band shifting using my protein
>and a small fragment DNA. The small fragment is about 20 bases, which is
>planned to be synthesized. For sure, the synthesis of a 20-mer of
>oligonucleotide is much cheaper than that of two strands to make a ds DNA.
>My question is if some methods can be used to synthesis such small ds DNA
>fragment (20bp) from the single strand oligonucleotide.
>Any suggestion will be appreciated.
I used to label my probe for gelshift in this way:
Synthesis of the 20mer of interest
Synthesis of a 4 (or 6mer)complementary to the 20mer.
Klenow reaction using the 20mer as a template and the 4mer as a primer.
Usually I use a 3-4 fold molar excess of primer. The radiolabeled nucleotide
can be changed according to the base composition of the template to obtain
the desired specific activity of the probe. I used this method to obtain
high specific activity probes.
The method is simple and even if you need two oligos, one of them is only a
4 or 6mer and therefore cheaper and if you want to save you can design
several oligos with the same end and use the same primer for all of them.
Check that you don't create an artificial binding site by gelshift.
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