I work using two different clones that are isogenic except for two point mutations. I obtain these using QIAGEN Maxiprep Kit, and quantify them using espectrofotometer and nanodrop. However, when I transfect equal amounts (mass, eg: 2ug) of each in two different wells, I get different levels of expression. I checked purity by espectrofotometer ratios and they are both pure. I also checked their mobility and aspect on agarose gels and everything is fine. I'm transfecting 293t and MOLT cells, and the problem happens in both cells.
The mutation is in a place that should not affect expression levels (not in any regulator/promoter region).
I need exactly equal expression in order to compare them on my experiments.
How do you know that the mutation doesn't affect protein folding or stability? It is very possible for these parameters to change with a point mutation, and thereby affect the expression level you achieve in your experiments.
If you run an "equal" amount on a agarose gel, do they still look like they have the same concentration? You may be getting incorrect readings from your spec due to subtle differences in the samples.
We refer to our nanodrop as an expensive doorstop... a qubit is better.
If I run equal amounts on agarose, they look just fine. I quantify the preps using a standart espectrofotometer, using nanodrop and also digest the prep and run on agarose gel using mass standarts, and everything is fine. But even using these three methods the expression is different. I really don't think it is a problem with protein stability, Actually it is a virus infectious clone, and the expression of all its proteins are lower in one prep than in another. The region is not a regulator region. And the most striking is that sometimes a pair of preps have WT expressing better than the mutant, and when I prepare new preps, this might change, and the mutant express better than the WT.
Two possible factors that could be at play here. First, perhaps you are getting different levels of endotoxin contamination in your maxipreps leading to altered transfection efficiency. Are you using a low endotoxin maxiprep kit? Second, some plasmids can be highly concatenated after purification, and concatenated plasmids transfect at lower efficiency. Check for this by running undigested plasmid on your agarose gel. You may need to linearize your plasmid prior to transfection.
Hi Luiza,
How did you solve the problem? What was the reason of your problem? Thanks
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