Hi all,
my lab has been using the SequalPrep Normalisation Plates from Invitrogen for a couple of years now, however we have never been able to get the expected concentration of 1-2ng/ul when using 20ul elution volume. We usually get a concentration in the range of 0.2-0.8ng/ul. The starting material we used is >250 ng amplicon per well. We have contacted the customer support before but it could not be explained why our concentration was so low.
I would just like to hear from others who have used the plates and what your experiences were and whether there any tips or tricks when performing the normalisation?
Thanks.
Have you tried using the optional elution method? We would elute rows a-d together and e-h together or you can follow this from the protocol.
optional sequential elution method is designed to sequentially elute multiple rows or columns using the same 20 μl of elution buffer to obtain higher amplicon concentrations. The amplicon concentrations will be additive as sequential wells are eluted. For example, dispense 20 μl of elution buffer into the first column (A1 –H1), mix well, and incubate for 5 minutes at room temperature. Then, simply move this column of elution buffer to the next column (A2–H2), and again incubate for 5 minutes. Continue this step to obtain your specific elution needs for the downstream application of choice.
Thanks Judith S Opp ,
my question is, would that not concentrate the DNA in the elution buffer?
Ideally I do not want to concentrate but I would like to elute more from the wells. At the moment, my research group has decided to go back to manual normalisation.
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