Hi i have a quiz for you guys.
i don't know how to solve this trick.
attached you ll find flow chart with my issue in dettails.
Briefly, i tried to purify a plasmid trough maxiprep (two different kits) no yield but the same culture in miniprep worked.
I tried another plasmid (same storage condition of bacterial batch) and the maxiprep was fine.
No clue what damn is going on.
Any option?
Hi there,
I think you should definitely scale up the miniprep purification for P1. A few minicolumns in parallel should be enough instead of trying to sort out the situation with MaxiPrep kits.
I need more than 1mg of plasmid.....is means almost 40-45 mini prep. Do you think its ok?
Well you need a lot so definitely need the adapted kit working. As the 2 you use are not up the function, go for a fully new one or one from an other company.
What's the size of your starter culture? My lab normally uses single colonies for any culture. We use an overnight starter culture of 3mL (used for mini prep), and scale up if needed (a maxi prep calls for 100-200mL), proving more time for growth.
Starting with an entire glycerol stock can give you heterogeneous DNA. Starting with too big of a culture for a single colony can also give you junk DNA.
You didn't indicate your elution volume. You could be seeing nothing from your maxi prep because your elution volume is too high or too low (900ug/uL is very concentrated).
You should check if you see your plasmid in your Maxiprep lysate by IPA precipitating a sample of it. If you see your plasmid in large amount, something is wrong with your Maxiprep purification as you lost your plasmid down the road. We routinely precipitate the samples throughout the prep with IPA at Biozilla when we have issues. You always get your answer this way. Good luck.
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