I work with Pontoniine shrimps (mostly Cuapetes). In the protocol it originally says I should use 150 µl of AE buffer in the last step, but some people say the less you use, the better (because the DNA is more concentrated then). I was advised using 50, but I forgot and used 100. Do you think it can effect the results much?
When I worked with other Palaemonidae, I was using 150 and PCR still worked, and the error rate was mostly done by different requirements on temperature, etc.
Thank you for any help.
The major effect of the volume of AE buffer used will be on the final concentration of DNA. Therefore, the volume of AE buffer (or any elution buffer) to use depends on the amount of tissue you are starting with and what concentration of DNA you need. Small amounts of tissue and high concentration of DNA = small volume of elution buffer, and vice versa. I have used as little as 25 ul of buffer when working with very small amounts of tissue. If your downstream application works with the 100 ul volume, then that is fine.
I think I had a small amount of DNA to work with (mostly shrimp eggs and tiny bits of tissue), but I had almost zero experiences and couldn't guess if it's a lot or not...I hope I didn't screw up too much and can still count with some results, because I don't have spare material. But the mistake must be somewhere else (too), last time not even a Positive control worked...
Thank you for the help!
Bara,
I was using 25 ul of AE buffer with 10-20 coral embryos, which in my experience is not a lot of tissue. If the concentration is too low, it is possible to concentrate your DNA using a column such as Zymo's Clean and Concentrator. And I agree that if the positive control didn't work, there is probably something else going wrong. Don't let it discourage you.
Cheers,
Lauren
Bara,
From what I've found, the recommendation in the protocol is only a generalisation, so you shouldn't worry too much about that - it would make more sense to work backwards from the requirements of the analytical assays you will use the DNA in, and decide whether you need high concentrations of DNA, or whether more eluted DNA at lower concentrations will work better.
I have no experience with the samples you're working with, but you may want to consider that in eluting DNA in larger volumes of buffer, you will also effectively reduce the concentration of any inhibitors that may have persisted through your extraction process.
There are probably other factors that you need to optimise, based on what you have said, but maybe you can achieve a compromise with regard to your question - elute in the lowest volume practicable to start with. You can re-dilute a partial volume from this if the DNA concentration is more than you need, or you think you are experiencing issues with inhibitors.
Good luck!
Yes Bara - you should be able to elute your DNA pellet in whatever volume of buffer you think is most suitable prior to using as template in pcr (as you can see from the different volumes people have indicated in their answers). Hence, if you elute in a more conservative/lower volume to start with, you will have a higher concentration of DNA. If you find that the concentration is more than sufficient for your requirements, you can dilute your DNA further do that you can do more pcr reactions with it.
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