6 replies to this topic

[perneseblue]

I have come to ask for the collective wisdom of Bioforum

Earlier this evening,  I got into a debate with a labmate of mine. We were discussing the merits of column based DNA purification method versus modified alkaline lysis method for BAC extraction.

While there were many point which we disagreed on, one point she raised had me thinking.

She claimed that column based BAC purification has less contaminating bacterial genomic DNA. The reasoning being that genomic DNA being longer than BAC DNA, has a stronger affinity to the column matrix. Thus when the DNA is eluted from the column, BAC DNA is preferentially eluted.

I on the other hand think that at 10kb and larger the binding affinity of a BAC and sheered genomic DNA would be no different. Both would bind and dissociate with equal efficiency to the column matrix.

What is really happening within the column?

This question could be answered by experiment. But that would take actual work and time - next week.

[hwtham]

I think we will always somehow get traces of genomic DNA in our eluted plasmid sample. Just that because the eluted sample contains majority of plasmid molecules, subsequent experiments show good results.

[bob1]

I think you are correct perneseblue, the size differentiation is based on charge of the DNA as far as I can tell, so BACs and sheared  genomic DNA should bind with equal efficiency.... however, if you are careful with the extraction there shouldn't be much sheared DNA.

[caltransplant]

Traces of genomic DNA will elute, but you won't get complete removal from the column.  Learned this one the hard way after trying to column clean genomic digests...

[Nephrite]

Hello :-)

I was just wondering where to post my question and I saw your discussion.

I work with RNA`s. Can you explain me how actually the step with Qiagen gDNA Eliminator spin column removes the gDNA from the sample? It is based on.....something like size-excluzion filtration, or...?

Thank you,

Nephrite

[bob1]

It will be based on the solubility of DNA and RNA at different pH I suspect.

[perneseblue]

It is due mainly to the pH of the buffers.The interaction between DNA, RNA and column matrix is pH and salt dependent.