The Gibson Assembly Mix is assembled on ice for obvious reasons, including not chewing back those ends too far. However, is there a suggestion to go into a machine pre-warmed to 50 degrees C or should the machine go from 25 to 50 with the samples in it? This will have something to do with balancing the denaturation rate of the exonuclease versus its activity at 50.
There seems to be lots of room for deviations. I pipet mine together at room temperature, mix them, and walk them over to a pre-temperated water bath. Done scores of them. They fail due to choosing the wrong sequence overlaps (G/C rich, secondary structures...). They're very robust against handling details.
Think about it -- it doesn't really matter if nuclease chews back 25 base pairs or 250. Pol will fill them back up when it's all said an done, as long as there's a single bp left double-stranded.
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