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Help Needed Finding Some Plasmid Prep Protocols

plasmid cell culture transfection plasmid prep

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2 replies to this topic

#1 Gandalf99

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Posted 28 May 2018 - 08:15 AM

My lab just received our own custom plasmid that we ordered in the form of a dry large scale prep (a small tube with just dry DNA in it). First thing i did was wrap the tube in aluminum foil and put it in the fridge.

 

Protocols:

  1. I need a protocol for resuspending the plasmid DNA for both use and long term storage (I believe we need TE buffer for this?).
  2. I need a protocol for transfection of HEK293 cells (mammalian) with our plasmid. Our cells are adherent type, growing in a media of DMEM+FBS+Glutamax.


#2 OldCloner

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Posted 03 July 2018 - 11:57 AM

I can help with #1.  If the plasmid was already in TE (or tris buffer without EDTA) when it was dried down all you would need to do is add sterile distilled water back to its original volume.  I'd check with the supplier to see if that is the case, and see what their instructions are for resuspension.

 

For the other you'll need someone with cell culture experience, sorry!



#3 bob1

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Posted 03 July 2018 - 01:12 PM

In addition to OldCloner's post, I would say to transform bacteria and make a glycerol stock of them so that you can do new preps of the plasmid whenever you need to.

 

As for transfection - any of the commercial transfection reagents work well, just follow the protocol supplied with the reagent. I have used Fugene and Lipofectamine (most of the forms) as well as TransIT with good success for all. HEK293 transfect easily, so you shouldn't have any problems.







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