Amplify retroviral vectors doesn't work from miniprep to midiprep - (Aug/15/2006 )

Hello ,

I am seeking for help on the amplification of retrovirus-producing vectors. I have 3 vectors for retrovirus production, which are pVSVG, pGag/Pol and pMIG-w. The plasmid DNAs of the 3 have been purified by someone long time ago (precipitated in isopropanol)and been kept in -20C for years (>5yrs). My recent work requires the production of retorvirus so I restored them and transformed it into competent TG1 trying to amplify more. 5ml of transformed bacterial culture have been miniprep (Qiagen) and RE digestion to confirm the DNA quality. Things are alright at this stage. The problem comes when I try to scale up the bacterial culture (5ml to 100ml) and extract DNAs using midiprep (Quagen). I usually have very poor yield from this step (no DNA can be seen when adding isopropanol; concentration far below 1ug/ul) although all the conditions are the same with the miniprep. (ps. both Qiagen kits are new and buffers are in good condition). Anybody has any clue what's wrong in my midiprep ?? any advice or experience shared are appreciated !!

I'm working with two different retrovirus-producing vectors systems. All low-copy plasmids. Which (as far as I know is often the case for these vectors) With Midi-prep I also got very little DNA (below 1µg/µl). Therefore I'm doing Maxi- or even Giga-Preps now. Using the corresponding kits from QIAGEN.

Even if the kit components and buffers are new, its better to check first if they are working. You can do that by doing midiprep for other known DNA. Finally in how much water/TE are you dissolving the pellet? Because if you dilute it too much ug/ul will be less. Check if you are not loosing the pellet while reconstituting during the last step as here it is not small microfuge tubes but bigger 50ml tubes!

Thanks to Jou and Calvin I will try to grow my bacteria in larger volume then try Maxi again. and Yes, it was a problem for me to identify the pellet when reconstituting in 50ml polyethlene tube. But I did aliquot it into microfuge tubes (though plenty of them) for better viewing of the pellet. In my poor yield of the plasmid DNA, I only dissolved the tiny pellet into 100ul of TE beffer...

Many viral vectors r low copy plasmids. U need to grow a single colony in 10ml for like 10-12 hrs and then use the 10ml to inoculate 500ml culture and grow overnight. This should give u much higher yield.

I have been getting over 1mg DNA for lentivectors using this method.

Good Luck !!!

Thank you scolix, I'll take your note !

Has anybody been using the traditional Cs/Cl method to purify retroviral vectors ? Does this give you higher DNA yield in low copy plasmid ?