Lately in our lab, some of the gels have problems like smear image or strange markers. We have changed all the components like SDS, Laemmli, SDS running buffer, marker, Acrylamide, sds tank (mini protean tetra cell) and everything. More than one person had this problem so it is not a personal mistake either. In the same tank, sometimes two out of four gels are in this condition. What can be the problem here?
Dear Dilara Nazlı Erdoğdu
Is the pH of samples after adding Laemmli buffer all right? Are they blue or orange? (generally: blue = good, orange = bad, low pH)
@Tomasz Fraczyk, yes we are dealing with this problem with a lot of samples with changing pH values.
It must be a handcast gel...
Is it possible to obtain a single pre-cast validated gel and run your marker along with your samples to simplify your troubleshooting? even if the pHs are not OK and as Dr.Tomasz Fraczyk indicated all samples prepared with fresh and adequate Laemmli seem blue prior to load, you can trust to prestained markers. With the correct and fresh running buffer, if the voltage (approx. 200V) is appropriately working and not so much heat generation was observed you may exclude the misapplication as you pinpointed. Additionally, besides pH, control and desalt your sample. But as I stated above, handcasting reagents must be checked and the troubleshooting of this issue should be handled from its beginning.
Emir
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