I am trying to isolate DNA using a CTAB based lysis buffer and a guanidine hydrochloride based binding buffer at a pH of 6.
Final concentrations:
guanidine hydrochloride 1.73M
potassium acetate at 0.37M.
If I use this combination, the DNA does not bind to the membrane. Instead, it ends up in the flow through.
Does anyone know how to solve this problem?
Second question: I have seen a set of binding and wash buffers that are based on guanidine thiocyanate rather than Gu HCl. Can one simply replace Gu SCN by Gu HCl using the same molarities? The original recipe is here: http://arbol.uniandes.edu.co/Archivos/Ivanova-Membrane-Based%20Protocol.pdf
It might be a problem with ethanol concentration. If the wash buffer is not made correctly you may be washing off DNA...
Hello Beata,
Thank you very much for your help.
The DNA is lost in the binding step, not the wash step. But you're right, in principle DNA could also disappear into flowthrough in the washing step. However, the solution from the washing step contains hardly any DNA after sodium acetate-ethanol precipitation and resuspension into AE buffer.
In contrast, the flowthrough from binding contains 400 ng/uL if I do sodium acetate-ethanol precipitation and elute in 80 uL warmed AE buffer.
Silke
When trying to hybridise single stranded DNA it is typically done at much higher ph ~8.5 so pH 6 may mitigate against this, though the column retention and stability may dictate the optimum pH. If you are using a silica column it will probably be unstable at pH8.5.. As for substitutinh HCl for SCN, I would be careful - SCN would increase the disorder of the DNA as it is a chaotrope. Whether this would be beneficial again depends on the type of column binding being used.
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