I'm trying to purify lentivirus packaging plasmids for future lentivirus production. I've heard the opinion, that simple maxi-prep yields DNA with endotoxins, which reduces the virus titration. To which point is this true? Have you had successful virus production while using maxi-prepped plasmids? What yield reduction should I expect, if I use maxi-prep, compared to CsCl purification? Will using "endotoxin-free" maxi-prep kit from Qiagen ( http://www.qiagen.com/us/products/catalog/sample-technologies/dna-sample-technologies/plasmid-dna/endofree-plasmid-kits/ ) do any improvement compared with plane maxi-prep kit?
If you're packaging in 293s, those are generally unfazed by endotoxin. There's no way there will be enough endotoxin in your prep to disturb a 293.
I also use normal (endotoxin-full?!) maxi kits and don't have problems. Nothing could tempt me into doing CsCl purification so I can't comment on whether it makes a difference. My suspicion is that even if it does, it still isn't worth the hassle, and as nobody else bothers it is more likely an enormous waste of time. if your unconcentrated titres are ~10^7 that is very normal and, with the option of concentrating by ultrafuge, far more than needed for almost all in vitro/ex vivo needs.
We use first Invitrogen Midi Prep kit for plasmid isolation. With high yield and purity; we have done successful plasmid isolation and virus production. Then for scale up we tried qiagen maxi prep but we couldn't overcome the low yield and purity problem. Then we use qiagen megaprep kit. And we saw it is much more efficient then maxi prep. Higher yield and better purity of plasmids.
Yes, a classic maxi-prep protocol can work for purifying lentivirus packaging plasmids, and it's a common method used in many labs. However, when preparing plasmids for lentivirus production, the quality of the DNA is crucial. High-quality, endotoxin-free plasmid DNA is required to achieve efficient transfection and high-titer virus production. Here are some considerations and tips for purifying packaging plasmids:
Considerations for Purifying Lentiviral Packaging Plasmids:
Enhanced Methods:
While classic maxi-prep kits can suffice, there are specialized kits and methods designed to enhance the purity and quality of plasmid DNA for sensitive applications like lentivirus production:
- Endotoxin-Free Maxi-Prep Kits: These kits are optimized to remove endotoxins during the purification process, which is crucial for achieving high transfection efficiency in mammalian cells.
- Anion-Exchange Chromatography: Some advanced plasmid purification systems use anion-exchange chromatography, which can yield higher-quality plasmid DNA compared to standard alkaline lysis methods.
- CsCl Gradient Centrifugation: Although more time-consuming and labor-intensive, cesium chloride (CsCl) gradient centrifugation can achieve high purity and supercoiled plasmid DNA. This method is less commonly used due to its complexity and the availability of high-quality commercial kits.
Tips for High-Titer Lentivirus Production:
- Optimize the Transfection Mix: The ratio of packaging plasmids to transfer vector can significantly impact virus production. Optimizing this ratio can enhance virus yield.
- Transfection Method: Use a transfection method that is highly efficient for your specific producer cell line (e.g., 293T cells). Lipid-based transfection reagents are commonly used, but electroporation might be preferred for some cell types.
- Culture Conditions: Healthy, actively dividing cells at the right confluency can significantly improve transfection efficiency and virus production.
In summary, while a classic maxi-prep can be used for purifying lentivirus packaging plasmids, opting for an endotoxin-free preparation and paying close attention to the quality and quantity of the purified DNA can significantly impact the success of your lentivirus production.
l Perhaps this protocol list can give us more information to help solve the problem.
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