I am wondering if anybody has opinions about using TAE Buffer vs the Buffer AE that comes with the Qiagen kit. We use the Qiagen DNA Stool Mini Kit to extract bacterial gDNA from stool samples, and the protocol says to elute the gDNA in Buffer AE, which is what we have been doing. But I have been doing some research, and Buffer AE only has 10mM Tris-Cl and 0.5mM EDTA, compared to TAE Buffer which has 40 mM Tris and 1mM EDTA. Should we switch and use TAE Buffer? We freeze the samples when we are not working with them. Would a higher concentration of EDTA potentially interfere with PCR reactions or other assays?
Dear Elizabeth
TAE buffer is a buffer containing a mixture of Tris base, acetic acid and EDTA.
TAE has a lower buffer capacity than TBE and can easily become exhausted, but linear, double stranded DNA runs faster in TAE.
In such cases, use of TBE buffer should be avoided as the borate of TBE buffer interacts with DNA and inhibits many enzymes (such as DNA ligases).
Overally, TAE Buffer is used effectively for separating fragments which are larger than 4000bp and is also used to separate super coiled DNA. Whereas TBE Buffer is effective for the separation of fragments between 1 and 3000bp in length. It provides an ionic solution in order to allow the current to pass through the water.
About your question, the recommended concentrations for TAE are:
40mM Tris, 20mM acetic acid, and 1mM EDTA.
But I used 0.1 mM EDTA in several works and find valuable results.
In both buffer EDTA was existed. EDTA is a chelating agent and has great affinity with matel ions and Mg-ion present in DNase as a cofactor and responsible for DNase action that degrade the DNA,here EDTA bind with Mg-ion and nullyfy the action of DNase.
Therefore, Its effects are only on DNAase and other metalo-enzymes and higher concentration of EDTA not affect DNA structure because EDTA have negative charge and there are repulsive forces between them.
Dont worry about it
Cheers
For DNA storage, you could use TE (Tris 10mM, EDTA 10mM, pH7.4) or water. If you use TE, in PCR reaction, you might have some inhibition, if you use to much volume of your sample.
yes, a higher concentration of EDTA could inhibit PCR assays.
TE is typically 10 mM Tris, 1 mM EDTA at pH 8.
It's quite common to use 10 mM Tris, 0.1 mM EDTA pH 8 to store DNA.
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