Hello all! Hope you are doing fine.
For the needs of a library preparation (Illumina sequencing of PCR products basically), I need to dry 100ng of each PCR product and then reconstitute it in 2µL of ddH2O overnight.
Thus my DNA (the PCR product) is still stored in the PCR mix.
My question is simple: Can I simply leave the PCR plates open for one day or two at room temperature until evaporation? Will the DNA evaporate with the PCR mix?
I am aware that precipitation methods exist etc but I'd like to avoid them...
Also, I'm interested in the theory behind this so I would be grateful if you could detail your answer (more than just "Yes go ahead!").
Thank you very much for your time, it's appreciated !
Noé
I won't recommend going for air dry with the PCR product that is still in the PCR mix. The reason is PCR mix contains lots of salts, leftovers dNTPs, and primers that will cause problems with the downstream process (in your case library preparation). For library preparation, high-quality DNA is required that won't be possible with air dry or speed vac. By high quality means the 260/230 ratio should be around 2. Moreover, with that many impurities, the nanodrop reading won't be good either.
The precipitation methods are not only useful to concentrate the DNA but also removes these impurities. Instead of precipitation, you can use a PCR purification kit that is fast.
Hi,
I do not know the specific method suggested by your library prep kit. I think you should use magnetic beads to isolate the PCR fragments then precipitate and wash them in alcohol 100% and 70% respectively. Then dissolve in NF H2O and proceed further. Just evaporating your PCR mix will leave salts behind. DNA will not evaporate. The above-mentioned process is pretty standard in library preparation.
Cheers.
Thanks Prabodh Kumar !
Ok, so assuming I'm doing a purification step before drying, I will get rid of all the PCR primers , dNTPs etc. And then I can just leave my purified PCR plates open at room temperature, is that right?
Thanks again for your help :)
When you have purified the product there is no need to dry. After precipitation with the beads and 100% ethanol, you'll need to wash with 70% ethanol to remove all impurities. Once that is done your sample will dry within 10-15 minutes as 70% ethanol evaporates in no time. Then, elute the bound DNA from the beads with NF water and then you are good to go to the next step. All the impurities including leftover dNTPs from the PCR will be cleared. If you do not have the DNA purification beads you might use the PCR purification kit. However, I have never used it. But that should work like Sayenta Bera mentions.
Thanks again Prabodh Kumar ,
the thing is my PCR products will be of different concentrations so I will quantify them and transfer equal molar amounts of products for each sample into a new plate (thus different volumes but same DNA quantity) and then I need to dry it and reconstitute it in 2µL of ddH20 to end up with equal volumes of equal concentrations of my PCR products for each sample.
Thus, I really need to dry them, so again do you think I can do that simply at room temperature?
Noé Barthelemy Ok, I understand that you will need equal volumes of equal concentrations of different PCR products. Now, you can use the method I previously described and bring all your different samples to equal concentrations by diluting them in NF or dd H2O, which I highly recommend.
However, I do not completely understand the problem you are facing. If you really want to dry your PCR products I will suggest using a speedvac vacuum concentrator or a sterile hot air oven set to room temp. I do not suggest drying it in room temperature in the open because it might take a long time and there might be a chance of contamination.
The best option is to consult the Library kit provider. I do not think you have to dry it because how would you measure it? Take your time with this, I know what a pain library preparation is :)
Prabodh Kumar
Very good advice, thanks a lot !
I'm probably go with the oven option and use the precipitation method if it fails!
Have a very good day Prabodh :)
You should do a PCR clean up before drying. You don't want to concentrate salts in that 2 ul. Place ur PCR product in PCR machine for 70 degrees for 30 mins and u should get it dried.
You can, but it will take a long time.
Do you have access to a vacuum centrifuge?
Just for clarification, I will clean and purify the Products before quantification and thus before drying, I forgot to mention it :)
Katie A Burnette , I don't !
I have a speedvac but it uses 1.5mL tubes only, so that doesn't help.
So you think that leaving the plates open at room temperature, in a clean space (to avoid contaminations) can do the job? Have you done it before? My protocol then relies on reconstituting the 100ng of DNA in 2µL of H2O to end up with equal concentrations of DNA for each individual and avoid most overrepresentation problems in the sequencing of the library. Thus, I'm very interested in knowing if air-drying is reliable (i.e that I won't loose too much DNA by evaporation etc). Is it any better in a 30°C oven, or just more risky ? Any details are welcome !
I'm very surprised to see how hard it is to find information about such a basic question online!
Thanks all for taking time to answer, I appreciate it!
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