Ligase free annealing question

EK someone at microsoft.net
Fri Jan 23 11:40:42 EST 2004


"redeamer" <alion85 at hotmail.com> wrote in message
news:a86fd891.0401230813.614821ab at posting.google.com...
> ....two complementary oligonucleotides containing AR binding sites
> were obtained from commercial provider (MWG AG BIOTECH, Germany). In
> addition to AR binding site oligonucleotides contained two
> 5'-overhanging deoxyguanosines for fill-in labeling of resulting
> oligonucleotide overhangs with radioactive alpha-[32P]dCTP using the
> Klenow fragment.
>
> Oligonucleotides AR-1 (10 microl) and AR-2 (10 microl) were mixed in
> equimolar amounts (1 nmol each) in 70 microl of ddH2O and 10 microl of
> 10x annealing buffer (500 mM TrisHCl pH 8.0, 100 mM MgCl2 and 1M
> NaCl). Annealing reaction was performed in PCR machine (5 min 95°C, 10
> min 70°C, 10 min 65°C, 10 min 60°, 10 min 55C°, 10 min 40°C).
> Resulting double-stranded oligonucleotide concentration was
> approximately 200 ng/microl.

Thanks a lot! How about annealing to a linearised vector with complementary
sticky ends? Would same buffer and say 50-fold molar excess of the insert
vs. vector be OK? The problem I guess is in the efficiency, because annealed
vector/insert product will be used to electroporate E.coli, and if the yield
of closed circle is too low, no colonies will show up...
-Emir





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