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phage434, on 28 May 2013 - 05:41 AM, said:
Most column based purification kits omit the ethanol in the wash buffer for shipment. The ethanol needs to be added before the wash buffer is used. Make absolutely certain your wash buffer has ethanol, or your DNA will be washed away.
You can trace where your DNA is going. You know there is a band of input DNA. Sample all of the flow through, wash, and elutions and run them on a gel. Somewhere, your DNA is hiding. You might want to try this with a DNA sample which is easier to produce -- the problem is likely not specific to your DNA sample.
You can trace where your DNA is going. You know there is a band of input DNA. Sample all of the flow through, wash, and elutions and run them on a gel. Somewhere, your DNA is hiding. You might want to try this with a DNA sample which is easier to produce -- the problem is likely not specific to your DNA sample.
ethanol has already added into wash buffer (my colleague did it). sorry for earlier misunderstanding.
How should i trace the hidden DNA? i don't understand clearly, should i load the binding solution and wash buffer trash onto the gel?
the generated band is not in a bright intensity. then i thought to dilute it in a less TE/water/elution volume. but, both PCR and gel clean up were still the same. not any progress revealed.
meanwhile, when i worked with 16S primer, things gone just right. i had no problems in a pcr purification stage.
