September 13, 2010 at 6:25 am #1209
When I do high throughput sequencing (generally for human DNA), I make up two mastermixes: one big mastermix for all my human reactions, and a small mastermix for my sequencing control (this is plasmid DNA). Both mastermixes use the same reagents, even the same primer, except the DNA is just different species and the reagent volumes in each mastermix are different, having been optimized for that DNA type. So for example the human mastermix adds 0.05ul of sequencing primer, while the plasmid mastermix adds 2ul of the same sequencing primer.
There has been times where my plasmid control has worked correctly but the rest of my reactions failed, or vice versa. I first think that I made a mistake during my mastermix setup for either DNA but can’t really tell since these are two different mastermixes, so my question is if it is possible to make a mastermix where human and plasmid DNAs can be sequenced using the same mastermix? This way I can at least rule out I made up the mastermix correctly or not. If yes, what are the reagent volumes I should use? .
My reagents are: sequencing buffer, DMSO, sequencing primer, sequencing dye, and DNA for both mastermixes, just in different volumes for each.
November 4, 2010 at 6:25 am #1210
Is there any reason why you can’t use the same master mix for both DNA’s. If the only thing that is different between the two mixes is the DNA then keep them the same. Use the human DNA conditions for your plasmid control. If the conditions work for the more difficult human DNA templates then they will work for the much easier plasmid.
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