[pmiguel]

what is the specific role of Hi-Di formamide?

Formamide is just the solvent in which the reaction products are resuspended after "cleaning-up" the reaction. "Hi-Di" is said to be of high quality -- we were told it isn't oxidized like other sources of (cheap) formamide would be.

Back before cycle sequencing was common for generating reaction products, it was critical to denature the products from the template strand before subjecting them to electrophoresis. But after denaturation formamide might have been added to slow the renaturation of products back to template strands.

That said, the main reason to use it now would be because it generally produces much more even signal electrokinetic injections. That is, prior to electrophoresis the samples must be moved into the polymer-filled capillaries. So the tips of the array are dipped into the wells of a 96- or 384-well plate in which the samples have been resuspended. Then a brief burst of high voltage electrophoresis occurs that moves a portion of the reaction into each capillary. This process is called "electrokinetic injection". Then the tips are move over to the buffer tray and normal electrophoresis commences.

Only a tiny percentage of the reaction actually moves into the capillaries. If you use pure water (18.3 M-ohm/cm) to resuspend the samples instead of formamide, much (~5x) higher signal results. However, this results from molecules farther from the tips of the capillaries electrophoresing in than occurs with formamide solvation. As a result shorter products will preferentially be injected because they have lower mass and will move more quickly in the applied voltage difference. So signal of the longer products suffers at the expense of the shorter products.

Formamide, probably just by limiting the strength of the local field, greatly ameliorates this issue. I would say this is the main reason that formamide is used to solvate samples these days.

--
Phillip

[quratulain]

please tell if any one knows that is it enough to exclude a gene on the basis of sequencing that gene in only one affected member of the autosomal recessive hearing loss family or do i need to do sequencing of the gene for all the affected members to exclude that particular gene in a family?

[GenoMax]

please tell if any one knows that is it enough to exclude a gene on the basis of sequencing that gene in only one affected member of the autosomal recessive hearing loss family or do i need to do sequencing of the gene for all the affected members to exclude that particular gene in a family?

Please create a new thread for this. People are likely to miss this question buried in a thread titled "sequencing protocol".