Hi all,
I'm attempting to clone a GC rich insert (500bp) into a vector that is approximately 5kb and also GC rich. After sequential digests (one enzyme works at 37 while the other works at 65 degrees Celsius), a 0.5% agarose gel reveals that the vector was efficiently cut as indicated by a 500bp shift down of the parent insert vector. Oddly, the 500 bp insert is barely visible. When blown out, the gel shows a smear near the 500 bp region. Is there a reason this is occurring? We are struggling to get any colonies to appear for diagnostic digests so any help would be appreciated.
Thank you!
The relative intensity of the 500bp band will of course be only ~10% of that of the vector band. So unless you have a very bright vector band, the small band is likely to be quite faint. Also small bands tend to diffuse more rapidly, so if there was a significant time lapse between running the gel and imaging it, that might be the explanation. But if this is an important clone you might wish to verify by sequence or PCR.
Hi there,
To go further in Michael's comment I would add the use of a more concentrated gel will help to get a thiner and therefore a more intense band. 0.5% is a very low concentration suitable for large DNA fragments. 2% agarose will be perfect to get a better resolution in the range of 100-500 bp.
Hi
I agree with D. Liger. You should use more concentrated gel. If the problem still exists, you can increase the time of the restriction enzyme digest.
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