How can I precipitae DNA in 96-well-plate? - (Jan/23/2008 )
Now I try to construct home-made DNA array. I cloned all target DNAs into T-vector and amplify them for spot onto nylon membrane. But now I have a problem about PCR product purification, I have target DNAs around 380 genes and I have to purify all of them in a few minute. So I would like to ask someone who have an experience about DNA precipitation in 96-well-plate. Is it any method that can use for this propose? There is a centrifuge that can be used for 96-well-plate in my lab but the speed of it very low (around 4000xg). So I think it not work if I use amonium acetate precipitation in this case. Someone help me please? Thank you very much for your help.
It will work. More problematic is removal and wash without disturbing the pellet. Typically this is done at very low speed with the plate upside down cushioned on paper towels. You might try with glycogen or pellet paint until you get it right.
Think hard about whether you really need to do this. Can you instead just spot the PCR product directly without purification? Are the primers unique?
Think hard about whether you really need to do this. Can you instead just spot the PCR product directly without purification? Are the primers unique?
Thank you for your kind suggestion.
Form you suggestion, that mean I should try precipitate the PCR product by glycogen and have to find out the optimal amount of glycogen for PCR product precipitation, right? I just wonder about the speed of centrifugation for nucleic acid precipitation. Trypically, I alway use the high speed (around 18,000xg) for this aim and never use the low speed. Is it work?
That you ask me about the PCR product can be spot directly without purification, I don't know it can be spot or not. Because this is the first time for me to spot the target DNAs onto nylon membrane and I have no experience about this technique. I still wonder about other material in PRC reaction will be effected on the other step (such as hybridization, and detection (I will use ECL detection technique)) or not? Sorry if I make you confuse in my english
. Thnak you for your kindness. I would certainly try spotting the PCR product directly. Do you plan to crosslink the spots with UV? If so, you can then wash the membrane to remove other material.
I'd STRONGLY recommend that you test all of this out, including the membrane spotting and ECL detection BEFORE doing it in 96 well plate format.
DNA can deffinitely be precipitated at lower speeds, it may just take longer. I'd mix the pcr product well with sodium acetate and isopropanol in the plate, freeze in the -80 for 30 minutes or so, and then run at the highest speed available for 30 minutes. I would do this the first few times with small amounts of glycogen or pellet paint as a co-precipitant so that I could see the pellets.
DNA precipitation can be done at 4000xg. When we do maxiprep according to qiagen's protocol, we do spin for 1 hr at 4000xg as that's the maximum we can go in a certain lab (there's no better centrifuge there and biosafety restrictions do not permit us do to it elsewhere). Works fine. In your case I would try first to do it in a couple of tubes at 4000xg, optimise the conditions (glycogen/pellet paint are helpfull) and then proceed with your 96-well plates. (to make sure: don't try your most precious plate first!).
Thank you for your kind suggestions I will try in a small scale first. And I will tell you after I get the result. 