Hello everyone I am running a pcr of some genomic DNA and it seems to amplify the genes I looking for fine however when I check the sample in the nanodrop I get a curve like in the picture 260/280 of around 1.80 but the 260/230 is like 0.5 can sombody tell me what is the problem with my sample? Is there a way of fixing it? can it still be used for sequencing?
Thank you
You will likely be fine to sequence. This is sometimes due to column purification (Qiagen) and sometimes due to oligo saccarides if you purify from agarose gel. Either way it should not interfere, alternatively you could purify again, and wash with EtOH (60%). If you need precise quant then pico green based method is preferred.
Dear Andres,
A low 260/230 ratio for DNA indicates contamination from phenolate ion, thiocyanates, and other organic compounds. It could also be because of carbohydrates and EDTA. If it is because of EDTA, then it could be a problem for sequencing since EDTA would chelate Mg(II) ions otherwise it should be fine..
But I think you should just go ahead and get it sequenced.
Best,
Jogender
You will likely be fine to sequence. This is sometimes due to column purification (Qiagen) and sometimes due to oligo saccarides if you purify from agarose gel. Either way it should not interfere, alternatively you could purify again, and wash with EtOH (60%). If you need precise quant then pico green based method is preferred.
A second column purification would save time and money. I would recommend it.
You have to purify your amplicons from agarose gel electrophoresis, I recommend you use columns from Zimo are so good and cheap, then you concentrate your amplicon, and then do your asymmetric PCR for sequencing and finally purify the sequence product by using Zimo's columns, then it is ready to use in the sequencer.
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