In my experience both excess salt and ionic byproducts (formic acid,
probably) generated from formamide cause lanes to curve. G50 purification
is effective in removing salts, but changing the deionized formamide stock
frequently is the only solution I have found to solving the formamide
derived problem.
Vahe Bedian
>We run a small sequencing core facility that accepts samples from various
>users. Usually
>the user has run the sequencing reaction and we run the samples on an
>ABI377, then
>post process the data and return it to the user.
>
>Lately we've been getting a lot of samples that case the lanes of the gel to
>be skewed
>toward the center of the set of samples on a gel. This seems to be a sample
>problem
>since, with mixed sets of samples on the same gel, the major skew will be
>toward the
>center of one group. Frequently the problem is very severe, requiring much
>manual editing
>and loss of data at the beginning of the run.
>
>I've suggested the cause is excess salt in the samples, resulting in
>increased conductivity
>within the wells of that set of samples, and so skewing the electric field
>and hence the lane
>pattern.
>
>Is that sensible? Does anyone have a better idea? What can be done to
>eliminate the
>problem?
>
>Thanks in advance.
>
>Mark J. Miller