I need to create a 105 bp dsDNA ( by denature the two oligos at 95 degrees for 5 min and than let it cool down slowly). Does anyone know if it will work with such long oligos ?
Should be fine, as the same thing happens at the end of every PCR reaction. Just use a thermal cycler to completely denature any secondary structures in the ssDNA (95 C for a minute or two), and then gradually lower the temperature back to room temperature.
Annealing doesn't happen at 95°C, at such temperature you denature the DNA.
yes, this will work. Don't forget to include some salt in the buffer for annealing. Typically something like 10 mM Tris pH8, 50 mM NaCl, 1 mM EDTA. I usually heat the tube in a dry block at 95C for 5 minutes, then pull the block out of the heater and let it cool slowly on the bench top.
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