Anyone know how can I precipitate PCR product 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.
-Nai_BC-
QUOTE (Nai_BC @ Jan 23 2008, 09:17 PM)
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.
How big is the PCR product? Have you run gels of the products? If the product is fairly clean, why not just transfer to the membrane? You'd be very unlucky if anything other than the target genes bound to the primers.
If you have to purify, you might need to use something like a magnetic bead system to capture the DNA and wash away everything else.
Good luck.
-swanny-
QUOTE (swanny @ Jan 23 2008, 03:11 PM)
QUOTE (Nai_BC @ Jan 23 2008, 09:17 PM)
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.
How big is the PCR product? Have you run gels of the products? If the product is fairly clean, why not just transfer to the membrane? You'd be very unlucky if anything other than the target genes bound to the primers.
If you have to purify, you might need to use something like a magnetic bead system to capture the DNA and wash away everything else.
Good luck.
Thank you for your kind suggestion.
My product size around 200-400 bp. After I check product on agarose gel, if them shown only one specific band, is it necessary to purify them? Because I would like to try to spot the target DNAs onto nylon membrane and I have no experience about this technique. I just 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. -Nai_BC-