I see reports in the literature that when two DNA single-strands are used to prepare a DNA double-stranded system, they first use a PCR machine to raise the temperature to 95°C, and then slowly cool to room temperature.
I am confused about that what exactly is this step? How long does it take to drop to room temperature? Is it possible to use a PCR machine to quickly cool to 4°C?
If you can answer my question, I would be very grateful.
If you have 2 single stranded dna molecules then the pcr machine heats the strands which breaks up any secondary structure like hairpin loops within each molecule. Then you let the block cool and as it cools through the annealing temperature of the 2 strands they anneal to each other. It is not necessary to cool to room temperature if the annealing temperatures of the strands are high temperatures. Once you have cooled to about 10c below the annealing temperature almost all of the strands will have annealed unless the concentration of single strands is very low. The reason for cooling slowly is to make sure that the longer homology regions between the 2 strands will anneal first compared with, for instance, short GC regions like CCCGGG annealing either between the 2 strands or even looping back within one strand to form a loop
If you don’t have a PCR machine, you can also mix the two strands in an Eppendorf tube, float the latter using a styrofoam floater in a beaker filled with boiling water and let the whole cool down to room temperature.
Hi Yuyan Liang,
Put your microtubes in a Thermoblock (or water-bath) at 95°C for 5min then immediately in crushed ice (or Freeze Block). Some reagents, when added, such as DMSO or formamide may also delay DNA hybridization for a few minutes.
Paul Rutland Thanks for your answer. In some reports, it mentions that they have to set some circles and set gradient cooling. What is the use of these operations?
Cycling seems pointless as it destroys the annealing done in the previous cycle so just one good denaturation then reannealing. the gradient cooling is to slow down the cooling as snap cooling will cause less annealing and leave more single stranded dna. Annealing happens quicker at warmer (but below melting temperature ) because thr molecules are moving around more so increasing their chance of meeting other molecules at higher temperatures.A slow reannealing step increases the DS yield because when most of the strands have annealed the remaining SS dna needs longer to find each other in the solution.
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