What is the best way to handle PCR fragments without damaging them?

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In summary, there is a possibility that sedimentation may have occurred with the PCR product sitting in the fridge for a week. Vortexing can potentially damage DNA if done for too long, so a short pulse (3-4 seconds) should be sufficient. However, it is also possible that the DNA may have started to degrade, but a week in the fridge should not cause significant degradation. It is generally recommended to store PCR reactions in the freezer for long-term storage. There is some disagreement among experts about the best storage method, with some recommending the fridge and others recommending the freezer. Vortexing should not damage PCR fragments unless done for too long, and it is advised to limit vortexing to 15 seconds or less. Shear
  • #1
rockind78
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I ran a PCR last week but did not get around to running the AGE until this week, so the PCR product sat for a roughly a week at -4*C. What are the odds that all my DNA is at the bottom or stuck to the sides? A friend of mine just suggested this to me, or I would have vortexed it before running the second AGE. Any thoughts?
 
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  • #2
Sedimentation migth probably happenned. It will not hurt to shake the reaction but vortexing can damage DNA if it is too long (more than 10 sec.). So a short pulse (3-4 sec.) should put everything back into solution.

In the other hand, the DNA migth have started to degrade. A week in the frigde is not that long and the degradation should be minor. It always best to store PCR reaction in the freezer (-20C).
 
  • #3
Originally posted by iansmith
It always best to store PCR reaction in the freezer (-20C).

I biochem professors would agree with that. Oddly enough, my molecular biology professor prefers the fridge. Thanks for the input though. That is just what I suspected.
 
  • #4
Do PCR fragments really settle?? I actually don't think so. I don't think so either that vortexing will damage your PCR fragments, how long are they? There really only is shear stress with genomic DNA, where flicking a tube with your fingers is adviced.
 
  • #5
Originally posted by Monique
Do PCR fragments really settle??

Without any movement it will but very slowly.

Originally posted by Monique
I don't think so either that vortexing will damage your PCR fragments, how long are they? There really only is shear stress with genomic DNA, where flicking a tube with your fingers is adviced.

When we order primers, the instruction to reconstitute them says to limit the vortexing to 15 secondes. The kit we use for genomic extraction also suggeste to vortex but to limit to 15 sec because shearing will start to occur.
 

1. How soon after collection can DNA settle?

The process of DNA settling can vary depending on factors such as temperature, humidity, and the type of sample collected. In general, it can take anywhere from a few minutes to several hours for DNA to settle.

2. Does the type of sample affect how fast DNA settles?

Yes, the type of sample can affect how fast DNA settles. For example, a liquid sample may settle faster than a solid sample. Additionally, the density and viscosity of the sample can also impact the settling time.

3. Can DNA settling be sped up or slowed down?

Yes, there are techniques that can be used to speed up or slow down the settling of DNA. For example, centrifugation can be used to separate DNA from other components in a sample and speed up the settling process. On the other hand, increasing the temperature or adding certain chemicals can slow down DNA settling.

4. How can I tell if DNA has settled?

There are a few ways to determine if DNA has settled. One method is to visually inspect the sample and look for a distinct layer of DNA at the bottom of the container. Another way is to use a spectrophotometer to measure the concentration of DNA in the sample and see if it has increased at the bottom of the container.

5. Why is it important for DNA to settle before analysis?

DNA settling is important for accurate analysis because it helps to separate the DNA from other components in a sample. This allows for better isolation and purification of the DNA, which is necessary for many types of analysis, such as PCR or sequencing. Without proper settling, the results of DNA analysis may be less reliable.

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