E-Gel Buffer Compatability

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The E-Gel manual recommends low salt buffers:

Important: Samples containing ≥50 mM NaCl, 100 mM KCl, 10 mM acetate ions, or 10 mM EDTA (i.e. certain restriction enzyme and PCR buffers) will cause loss of resolution on E-Gel® agarose gels. To obtain the best results, dilute samples which contain high salt levels 2- to 20-fold.

NEB Buffer 4 contains very high acetate concentrations:

1X NEBuffer 4:
20 mM Tris-acetate
50 mM potassium acetate
10 mM Magnesium Acetate
 1 mM Dithiothreitol
pH 7.9 @ 25°C


Whereas buffer 2 does not:

1X NEBuffer 2:
10 mM Tris-HCl
50 mM NaCl
10 mM MgCl2
 1 mM Dithiothreitol
pH 7.9 @ 25°C


Ligation buffer is also acceptable:

1X T4 DNA Ligase Reaction Buffer:
50 mM Tris-HCl
10 mM MgCl2
 1 mM ATP
10 mM Dithiothreitol
pH 7.5 @ 25°C


The symptom is extreme band smearing of gel bands.

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