Ligase free annealing question

redeamer alion85 at hotmail.com
Fri Jan 23 11:13:10 EST 2004


....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.



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