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Problem with Maxi Prep


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#1 altobarn

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Posted 10 May 2009 - 11:05 AM

I have constructed some recombinant plasmids and after miniprep they showed correct bands. However, when I planned to proceed to Maxi Prep I have seen some extra bands and I feel uncertain to proceed to Luciferase Assay. Could that possible because of I took the sample directly from Glycerol stock and put into LB medium without plating on LB + Amp plate first?

Anyone has a clue what went wrong with my maxiprep?

Thanks

-Altobarn-

#2 Vini

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Posted 10 May 2009 - 09:55 PM

I have constructed some recombinant plasmids and after miniprep they showed correct bands. However, when I planned to proceed to Maxi Prep I have seen some extra bands and I feel uncertain to proceed to Luciferase Assay. Could that possible because of I took the sample directly from Glycerol stock and put into LB medium without plating on LB + Amp plate first?

Anyone has a clue what went wrong with my maxiprep?

Thanks

-Altobarn-


Was your LB medium not supplemented with amp?

#3 altobarn

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Posted 10 May 2009 - 11:32 PM

As far as I concern, Yes! I have added amp to the LM medium. I'm suspecting the toxicity caused my glycerol or cross contamination caused the extra bands. Could it possible if the plasmid was too concentrated for enzyme test digestion and caused the extra bands as the plasmid was from maxiprep?

#4 Vini

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Posted 11 May 2009 - 04:03 AM

As far as I concern, Yes! I have added amp to the LM medium. I'm suspecting the toxicity caused my glycerol or cross contamination caused the extra bands. Could it possible if the plasmid was too concentrated for enzyme test digestion and caused the extra bands as the plasmid was from maxiprep?



well, i too thought about that. but if that is the case, it should be easy to judge. what is the size of this extra band that u r getting n tht of ur vector backbone? maybe u can enclose a labeled gel pic. of the prep.




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