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midiprep plasmids not expressing


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#1 ELISA DO LITTLE

ELISA DO LITTLE

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Posted 20 March 2011 - 10:46 PM

I used to be really crappy at midipreps. I use the qiaggen kits. My problem before was low yield. I solved this problem by precipitating my DNA in small epis rather than a large tube because our tubes are awkward and break in our centrifuge at high speeds anyway. I get good pellets and really good yields now using many small epis and a microcentrifuge. I have taken to removing the residual ethanol and leaving the pellets to air dry. Sometimes even overnight. The pellets are hard to resuspend but I will resuspend them with warm water and vortexing and they go back into solution because when I nanospec them I will get 1 ug/ul typically (250ul). When I transfect these plasmid preps, however, I do not get expression. What could be the issue here. These are established plasmids that I am just making more of.

#2 pito

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Posted 21 March 2011 - 09:17 AM

Its possible you are simply destroying your DNA... Overnight drying and vertoxing... thats not good.
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

#3 pDNA

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Posted 21 March 2011 - 09:56 AM

have you ever checked your plasmids on an agarose-gel before transfection?
Never ever trust an spectrophotometer when dealing with DNA (1st law of molecular biology!) ...only the agarose gel tells you the truth!
Maybe it is just genomic DNA you purified ...who knows ...or just sheared plasmid DNA that you broke in 10000000 pieces?

I would not dry the DNA overnight ...thats just to much ...15 mins should be more than enough! Overdried DNA is hard to solubilize. #
And if you don't feel comfortable with the Qiagen Midi (i don't like em either) ...try another one (e.g. for transfection grade plasmids the Machery&Nagel NucleoBond Xtra Midi EF with the Finializer is good choice!)

Good luck!
Regards,
p




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